CN103732743B - MPHOSPH1 peptides and the vaccine comprising them - Google Patents

MPHOSPH1 peptides and the vaccine comprising them Download PDF

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CN103732743B
CN103732743B CN201280039507.7A CN201280039507A CN103732743B CN 103732743 B CN103732743 B CN 103732743B CN 201280039507 A CN201280039507 A CN 201280039507A CN 103732743 B CN103732743 B CN 103732743B
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peptide
present
mphosph1
ctl
cancer
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CN103732743A (en
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角田卓也
大泽龙司
吉村祥子
渡边朝久
中村佑辅
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Oncotherapy Science Inc
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Abstract

As discussed in more detail in the application, the detached epitope peptide derived from MPHOSPH1 combines HLA antigens inducing cytotoxic T lymphocytes (CTL), is consequently adapted in cancer immunotherapy, more specifically used in the linguistic context of cancer vaccine.The peptide of the present invention covers aminoacid sequence derived from above-mentioned MPHOSPH1, and its modified forms, wherein replace, lack, insert or with the addition of one, two or several aminoacid, so long as modified forms retain needed for original series CTL inducibilities.Further, it would be desirable to provide the polynucleotide of any of above peptide of coding, and the medicine agent or pharmaceutical composition comprising any of above peptide or polynucleotide.The peptide of the present invention, polynucleotide and medicine agent or pharmaceutical composition are particularly useful in cancer and tumor, including the treatment and/or prevention of such as bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.

Description

MPHOSPH1 peptides and the vaccine comprising them
Technical field
The present invention relates to bio-science field, is in particular field of cancer.Specifically, the present invention relates to making For the useful new peptide of cancer vaccine, and for treatment and/or the medicine of prophylaxis of tumours.
Priority
This application claims the rights and interests of U.S. Provisional Application 61/522,991 that August in 2011 is submitted on the 12nd, by quote by Entire contents are expressly incorporated herein.
Background technology
It has been proved that recognizable MHC (MHC) the I quasi-molecules of cytotoxic T lymphocyte (CTL) Epitope peptide derived from the tumor associated antigen (TAA) of upper appearance, then kills tumor cell.From melanoma antigen (MAGE) family Race has been found, and people mainly have discovered that many other TAA (NPL1, Boon T, Int J by immunology means Cancer 1993 May 8,54(2):177-80;NPL2,Boon T&van der Bruggen P,J Exp Med 1996 Mar 1,183(3):725-9).Some in these TAA currently receive clinical development as immunotherapeutic targets.
Favourable TAA is indispensable for the amplification and survival of cancerous cell.Such TAA is used as immunity The target for the treatment of, can farthest reduce the risk that widely known carcinoma cell immunization is escaped.Carcinoma cell immunization is escaped can The caused TAA owing to the immunoselection (therapeutically driven immune selection) for treating driving Delete, be mutated or lower.Therefore, it is possible to induce the identification of the new TAA of strength and specific anti-tumor immune response ensure that Further develop, therefore for all kinds cancer peptide vaccine vaccination strategies clinical practice well afoot (NPL3, Harris CC,J Natl Cancer Inst 1996 Oct 16,88(20):1442-55;NPL4,Butterfield LH et al.,Cancer Res 1999 Jul 1,59(13):3134-42;NPL5,Vissers JL et al.,Cancer Res 1999 Nov1,59(21):5554-9;NPL6,van der Burg SH et al.,J Immunol 1996 May 1,156 (9):3308-14;NPL7,Tanaka F et al.,Cancer Res 1997 Oct 15,57(20):4465-8;NPL8, Fujie T et al.,Int J Cancer 1999 Jan 18,80(2):169-72;NPL9,Kikuchi M et al., Int J Cancer 1999 May 5,81(3):459-66;NPL10,Oiso M et al.,Int J Cancer 1999 May 5,81(3):387-94).So far, had the several reports that clinical trial is carried out using these TAA derived peptide.No Good fortune, the cancer vaccine test for currently carrying out much only observe relatively low objective response rate (NPL11, Belli F et al.,J Clin Oncol 2002 Oct 15,20(20):4169-80;NPL12,Coulie PG et al.,Immunol Rev 2002 Oct,188:33-42;NPL13,Rosenberg SA et al.,Nat Med 2004 Sep,10(9):909- 15).Therefore, yet suffering from identification in this area can be used as the needs of the new TAA of immunization therapy target.
MPHOSPH1 (M- phase phosphoproteins 1；GenBank Accession No.NM_016195 and NP_057279, SEQ ID NO:125 and 126) original is accredited as in G2/M transition by one of protein of pecific phosphorylation, and it is characterized as being anode Driving protein relative protein (plus-end-directed kinesin related protein) (NPL14, the Abaza of guiding A et al.,J Biol Chem 2003,278:27844-52).More specifically, previously it has been reported that MPHOSPH1 is a kind of The molecular motor (plus-end-directed molecular motor) that anode is oriented to, it plays the part of important in cytokinesiss Role, and in HeLa cells after cleaving during the phase to telophase of cell division spindle mesozone accumulation (NPL14, Abaza A et al.,J Biol Chem 2003,278:27844-52;NPL15,Kamimoto T et al.,J Biol Chem 2001,276:37520-8).Gene table in genome range cDNA microarray of the use containing 23040 genes Up to analysis of spectrum during, it is found that MPHOSPH1 is a kind of recruit (NPL16, Kanehira M et raised in bladder cancer al.,Cancer Res.2007 Apr 1;67(7):3276-85.;PTL1,WO2006/085684).Additionally, passing through Northern engram analysis, it is found that MPHOSPH1 gene outcomes are confined to testis, and be not present in normal life organ.
Some peptides with cytotoxic T lymphocyte (CTL) inducibility derived from MPHOSPH1 had previously been identified Fragment ((PTL2, WO2008/047473).These fragments show energy of the induction for the CTL of the cell stimulated with association fragments of peptides Power.However, previous research not can confirm that whether these fragments of peptides have induction targeted expression MPHOSPH1 genes and HLA-A2 The ability of the CTL of the tumor cell of antigen.
Reference list
Patent documentation
[PTL1]WO2006/085684
[PTL2]WO2008/047473
Non-patent literature
[NPL1]Boon T,Int J Cancer 1993 May 8,54(2):177-80
[NPL2]Boon T&van der Bruggen P,J Exp Med 1996 Mar 1,183(3):725-9
[NPL3]Harris CC,J Natl Cancer Inst 1996 Oct 16,88(20):1442-55
[NPL4]Butterfield LH et al.,Cancer Res 1999 Jul 1,59(13):3134-42
[NPL5]Vissers JL et al.,Cancer Res 1999 Nov 1,59(21):5554-9
[NPL6]van der Burg SH et al.,J Immunol 1996 May 1,156(9):3308-14
[NPL7]Tanaka F et al.,Cancer Res 1997 Oct 15,57(20):4465-8
[NPL8]Fujie T et al.,Int J Cancer 1999 Jan 18,80(2):169-72
[NPL9]Kikuchi M et al.,Int J Cancer 1999 May 5,81(3):459-66
[NPL10]Oiso M et al.,Int J Cancer 1999 May 5,81(3):387-94
[NPL11]Belli F et al.,J Clin Oncol 2002 Oct 15,20(20):4169-80
[NPL12]Coulie PG et al.,Immunol Rev 2002 Oct,188:33-42
[NPL13]Rosenberg SA et al.,Nat Med 2004 Sep,10(9):909-15
[NPL14]Abaza A et al.,J Biol Chem 2003,278:27844-52.
[NPL15]Kamimoto T et al.,J Biol Chem 2001,276:37520-8
[NPL16]Kanehira M et al.,Cancer Res.2007 Apr 1;67(7):3276-85.
Content of the invention
The present invention is based at least partially on can be used as the discovery of the new peptide of the suitable targets of immunotherapy.Due to TAA general It is perceived by the immune system for " itself " and therefore usually without immunogenicity, being the discovery that for suitable targets is extremely important.As above Described, MPHOSPH1 (such as SEQ ID NO:126, by GenBank Accession No.NM_016195 (SEQ ID NO: 125) gene code) have been identified as raising in cancer, the cancer includes but is not limited to bladder cancer, breast carcinoma, palace Neck cancer, cholangiocellular carcinoma, chronic granulocytic leukemia (CML), colorectal cancer, gastric cancer, nonsmall-cell lung cancer (NSCLC), pouring Bar tumor, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.Therefore, the present invention focuses on MPHOSPH1 as suitable cancer The candidate of disease/tumour immunotherapy target, can more specifically serve as the new of suitable immunotherapy target MPHOSPH1 epitope peptides.
For this purpose it is proposed, this invention address that, it is devoted to identifying have at least in part in the peptide derived from MPHOSPH1 Specificity epitope peptide of the induction for the ability of the CTL of MPHOSPH1.As further discussed in detail, using being derived from The HLA-A*0201 associativities candidate peptide of MPHOSPH1 stimulates the PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) obtained from healthy donors.So CTL system with specific cytotoxicity for HLA-A2 positive target cell through every kind of candidate peptide impulse is established afterwards.This Result in text proves that these peptides are the strong and specific immunne response of the inducible cell for expression MPHOSPH1 HLA-A2 restricted type epitope peptides.It is strongly immunogenic that these results point out MPHOSPH1 to have, and its epi-position is cancer/tumor Effective target of immunotherapy.
Therefore, it is an object of the present invention to provide detached peptide, which combines HLA antigens and induces CTL, wherein described peptide Including MPHOSPH1 (SEQ ID NO:126) immunologic competence fragment.These peptides can be used to external or in-vitro inducing CTL, Or for being administered to experimenter to induce the immunne response for cancer, the example of cancer includes but is not limited to bladder cancer, breast Adenocarcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and Soft tissue neoplasms.
The length of the peptide of the present invention is typically less than 15,14,13,12,11, or 10 aminoacid.The preferred peptide of the present invention It is nonapeptide or decapeptide.Particularly preferred peptide has selected from SEQ ID NO:5,14,64,73,77,79,97,103 and 120 amino Acid sequence, because these peptides show in conjunction with HLA-A2 antigens and can induce CTL.
Therefore, in some embodiments, peptide of the invention is that length is less than 15,14,13,12,11, or 10 amino Acid, have be selected from SEQ ID NO:The peptide of 5,14,64,73,77,79,97,103 and 120 aminoacid sequence.Typical real Apply in scheme, the peptide of the present invention is that have selected from SEQ ID NO:5,14,64,73,77,79,97,103 and 120 aminoacid sequence The nonapeptide or decapeptide of row.Additionally, as proved herein, with SEQ ID NO:The peptide of 120 aminoacid sequence is confirmed can The CTL of the tumor cell of induction targeted expression MPHOSPH1 and HLA-A2 antigen.Therefore, in preferred embodiments, this Bright peptide is with SEQ ID NO:The peptide of 120 aminoacid sequence.
When the present invention peptide in vitro, in vitro or when contacting antigen-presenting cell (APC) in vivo, by with APC on HLA- A2 knots are incorporated as being rendered on APC with the complex of HLA-A2 antigens.Or, the peptide of the present invention can be taken in by APC, It is processed to be by selected from SEQ ID NO in APC:5,14,64,73,77,79,97,103 and 120 aminoacid sequence is constituted Fragment, and as being rendered on APC with the complex of HLA-A2 antigens.As a result, the CTL of such peptide specific is induced Out, and such CTL be considered the present invention key element.
Present invention further contemplates that modified peptides, they have selected from SEQ ID NO:5,14,64,73,77,79,97,103 Hes Replace, lack, insert or with the addition of one, the aminoacid sequence of two or several aminoacid in 120 aminoacid sequence, as long as The modified peptides retain the CTL inducibilities being equal to original unmodified peptide.For this purpose it is proposed, the present invention provides length is less than 15, The detached peptide of 14,13,12,11 or 10 aminoacid, its have CTL inducibilities and comprising the aminoacid sequence being selected from the group Row：
I () is being selected from SEQ ID NO:5th, 1,2 or several aminoacid are instead of in 14 and 64 aminoacid sequence Aminoacid sequence, and
(ii) aminoacid sequence (i), the wherein aminoacid sequence have one or both of following features：
A second aminoacid of N-terminal of () described SEQ ID NO is selected from Leucine and methionine；With
B the C-terminal aminoacid of () described SEQ ID NO is selected from L-Valine and Leucine.
Additionally, the present invention also provides detached peptide of the length less than 15,14,13,12 or 11 aminoacid, which has CTL Inducibility and comprising the aminoacid sequence being selected from the group：
(i ') is being selected from SEQ ID NO:Instead of in 73,77,79,97,103 and 120 aminoacid sequence 1,2 or The aminoacid sequence of several aminoacid, and
The aminoacid sequence of (ii ') (i '), the wherein aminoacid sequence have one or both of following features：
A second aminoacid of N-terminal of () described SEQ ID NO is selected from Leucine and methionine；With
B the C-terminal aminoacid of () described SEQ ID NO is selected from L-Valine and Leucine.
As demonstrated herein, such peptide may with APC on HLA-A2 antigen bindings and as with HLA-A2 antigens Complex be rendered on APC.Or, when these peptides are contacted with APC, can be taken in by APC, be processed in APC It is by selected from (i), (ii) fragment of the aminoacid sequence composition of (i ') and (ii '), and as the complex with HLA-A2 antigens It is rendered on APC.As a result, the CTL of such peptide specific is induced out, and such CTL is considered the present invention's Key element.
Present invention also contemplates that the detached polynucleotide of the peptide of any present invention of coding.These polynucleotide can be used to lure Lead or prepare the APC with CTL inducibilities.As the peptide of the above-mentioned present invention, such APC can be administered to experimenter To induce the immunne response for cancer.
When experimenter is administered to, the peptide of the present invention is rendered on the surface of APC, to induce targeting corresponding peptides CTL.Therefore, it is an object of the present invention to provide compositionss or agent for inducing CTL, which includes one or more The there is provided peptide of invention or polynucleotide, for inducing APC and/or CTL.Such compositionss or agent can be also used for It is selected from following purpose one or more：Treating cancer, prophylaxis of cancer, and prevent the recurrence after operation of cancer.Target cancers Example includes but is not limited to bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymph Tumor, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.Therefore, a further object of the present invention is to provide for treating cancer And/or the medicine agent or pharmaceutical composition of prophylaxis of cancer, such medicine agent or compositionss are formulated as comprising one kind Or the peptide or polynucleotide of multiple present invention, as the replacement or supplement of the peptide or polynucleotide of the present invention, the medicine of the present invention Agent or pharmaceutical composition can have the APC or allochthon of any peptide of the invention as active component comprising presentation.
The peptide or polynucleotide of the present invention can be used to induce the complex for presenting HLA antigens and peptide herein on the surface APC, for example by make APC with the present invention peptide contact or by coding the present invention peptide polynucleotide importing APC in.This The APC of sample has the ability that induction specific recognition presents the CTL of the cell of target peptide in its surface, and can be used in cancer Immunization therapy.Therefore, the method that the present invention covers the APC that there is CTL inducibilities for induction, and obtained by such method The APC for obtaining.
Additionally, present invention also contemplates that agent or compositionss for inducing APC, such agent or compositionss include The peptide or polynucleotide of any present invention.
It is also an object of the present invention to provide for the method for inducing CTL, such method is included CD8 positive T cells The step of co-culturing with the APC or allochthon of the peptide for presenting the present invention in its surface, or import two φt cell receptors of coding (TCR) polynucleotide of subunit, or the step of encode multiple polynucleotide of each TCR subunit, wherein described TCR can be in conjunction with the peptide of the present invention presented on cell surface and the complex of HLA antigens.The CTL obtained by such method Treating cancer and/or prophylaxis of cancer is can be used to, the example of cancer includes but is not limited to bladder cancer, breast carcinoma, cervical cancer, bile duct Cell carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.
The present invention's it is also an object that provide detached APC, which presents HLA antigens and the present invention's on the surface The complex of antibody.The present invention also provides the detached CTL of the peptide of the targeting present invention.These APC and CTL are in cancer immunotherapy Linguistic context in useful.
It is a still further object of the present invention to provide should for being directed to the immunity of cancer in experimenter's Immune inducing in vivo in need The method that answers, methods described include that the step of applying agent or compositionss, the agent or compositionss include at least one It is selected from following component：The peptide of the present invention, the polynucleotide for encoding such peptide, present the allochthon or APC that have such peptide and Identification presents the CTL of the cell for having such peptide in its surface.
The application of the present invention extends to multiple related to MPHOSPH1 overexpression, or the disease for coming from MPHOSPH1 overexpression Any one of, such as cancer, the example of cancer include but is not limited to bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.
More specifically, the present invention provides following：
[1] the detached peptide of following (a) or (b)：
A () is comprising selected from SEQ ID NO:The peptide of 5,14,64,73,77,79,97,103 and 120 aminoacid sequence；
B () is included in selected from SEQ ID NO:Take in 5,14,64,73,77,79,97,103 and 120 aminoacid sequence Generation, disappearance, the peptide of the aminoacid sequence of 1,2 or several aminoacid of insertion and/or interpolation, and wherein the peptide has cytotoxic T Lymphocyte (CTL) inducibility,
[2] [1] detached peptide, the wherein peptide have one or both of following features：
A () is selected from SEQ ID NO:The N-terminal of 5,14,64,73,77,79,97,103 and 120 aminoacid sequence second Aminoacid is selected from Leucine and methionine；With
B () is selected from SEQ ID NO:The C-terminal aminoacid of 5,14,64,73,77,79,97,103 and 120 aminoacid sequence Selected from L-Valine and Leucine,
[3] the detached peptide of [1] or [2], wherein described peptide is nonapeptide or decapeptide,
[4] a kind of detached polynucleotide, its encode the peptide of any one of [1]-[3]；
[5] a kind of compositionss for inducing CTL, wherein described compositionss are comprising arbitrary in one or more [1]-[3] The peptide of item, or the polynucleotide of one or more [4],
[6] a kind of compositionss of the APC for induction with CTL inducibilities, wherein described compositionss comprising a kind of or The peptide of any one of multiple [1]-[3], or the polynucleotide of one or more [4],
[7] a kind of pharmaceutical composition, its include at least one active component being selected from the group：
The peptide of any one of one or more of (a) [1]-[3],
The polynucleotide of the peptide of any one of (b) one or more coding [1]-[3]；
C one or more of () presents in its surface the peptide of any one of [1]-[3] and the complex of HLA antigens APC or allochthon；With
D () one or more identification is presented in its surface the peptide of any one of [1]-[3] and the complex of HLA antigens Cell CTL,
[8] pharmaceutical composition of [7], for being used for treatment and/or prophylaxis of cancer in experimenter, or induction is for cancer Immunne response,
[9] pharmaceutical composition of [7] or [8], wherein described pharmaceutical composition are formulated as being applied to HLA antigens for HLA- The experimenter of A2,
[10] a kind of method of the antigen-presenting cell (APC) for induction with CTL inducibilities, methods described include The step of being selected from the group：
A () contacts APC and the peptide of any one of [1]-[3] in vitro or in vivo, and
B () will encode the polynucleotide of the peptide of any one of [1]-[3] and import APC；
[11] a kind of method for inducing CTL, methods described include the step of being selected from the group：
A CD8 positive T cells are had HLA antigens compound with the peptide of any one of [1]-[3] with presenting in its surface by () The APC of thing is co-cultured；
B CD8 positive T cells are had HLA antigens compound with the peptide of any one of [1]-[3] with presenting in its surface by () The allochthon of thing is co-cultured；With
C () imports polynucleotide of two φt cell receptor (TCR) subunits of coding in CD8 positive T cells, or Multiple polynucleotide of each TCR subunit are encoded, wherein described TCR can resist in conjunction with the HLA presented on cell surface The former complex with the peptide of any one of [1]-[3]；
[12] a kind of detached APC, the APC present the peptide of HLA antigens and any one of [1]-[3] in its surface Complex；
[13] APC of [12], its are induced by the method for [10],
[14] a kind of detached CTL, its are recognized and are presented the peptide for having HLA antigens and any one of [1]-[3] in its surface Complex cell,
[15] CTL of [14], wherein described CTL are induced by the method for [11],
[16] method of the cancer in a kind for the treatment of and/or prevention experimenter, wherein methods described includes applying experimenter With pharmacy effective dose
The peptide of any one of one or more of (a) [1]-[3]；
The polynucleotide of the peptide of any one of (b) one or more coding [1]-[3]；
C one or more of () presents in its surface the peptide of any one of [1]-[3] and the complex of HLA antigens APC or allochthon；Or
D () one or more identification is presented in its surface the peptide of any one of [1]-[3] and the complex of HLA antigens Cell CTL
The step of,
[17] a kind of method of the induction for the immunne response of cancer in experimenter in need, methods described includes right The step of experimenter applies the compositionss of the polynucleotide of peptide or coding for said peptides comprising any one of [1]-[3],
[18] antibody for the peptide of any one of [1]-[3] or its immunologic competence fragment,
[19] a kind of carrier, its include the nucleotide sequence of the peptide of any one of coding [1]-[3]；
[20] a kind of host cell, its carrier conversion or transfection through [19]；；With
[21] a kind of diagnostic kit, peptide which includes any one of [1]-[3], the polynucleotide of [4] or [18] anti- Body.
When connection with figures and embodiment read detailed description below, the purpose of the present invention and feature will become more to show And be clear to.It should be appreciated that brief summary of the invention and following detailed description above all only proposes exemplary embodiment, and The replaceable embodiment of other of the invention or of the invention is not construed as limiting.Especially, although just some concrete herein Embodiment describe the present invention, it should be understood that these descriptions are Illustrative for the purpose of the present invention, do not understand It is interpreted into limitation of the present invention.On the premise of without departing substantially from the spirit and scope of the present invention as described by appended claims Those skilled in the art will readily appreciate that multiple modifications and application of the present invention.Similarly, other purposes of the invention, spy Levy, benefit and advantage are that summary from there and particular described below are easily envisaged that, and are this Art personnel can be with obvious.And according to content above and therefrom can with reference to the embodiment, data, accompanying drawing that encloses The content that rationally infers, or further consider list of references cited herein, such purpose, feature, benefit and advantage It is readily apparent that.
Brief description
Those skilled in the art are in the Brief Description Of Drawings for considering hereafter and to of the invention and its preferred embodiment The application of various aspects of the invention is clearly learnt after detailed description.
[Fig. 1] Fig. 1 is made up of one group of photo (a)-(j), is described and is shown to being entered with the CTL of the inducing peptide for being derived from MPHOSPH1 The result that capable IFN-γ ELISPOT is determined.With MPHOSPH1-A02-9-850 (SEQ ID NO:5) (a) stimulate No. 7 Hole, use MPHOSPH1-A02-9-129 (SEQ ID NO:14) (b) stimulates No. 5 holes, MPHOSPH1-A02-9-846 (SEQ are used ID NO:64) (c) stimulates No. 5 holes, MPHOSPH1-A02-10-460 (SEQ ID NO are used:73) No. 2 holes, the use that (d) stimulates MPHOSPH1-A02-10-770(SEQ ID NO:77) (e) stimulates No. 1 hole, MPHOSPH1-A02-10-407 (SEQ ID are used NO:79) (f) stimulates No. 1 hole, MPHOSPH1-A02-10-923 (SEQ ID NO are used:97) No. 4 holes, the use that (g) stimulates MPHOSPH1-A02-10-1484(SEQ ID NO:103) (h) stimulates No. 5 holes and with MPHOSPH1-A02-10-282 (SEQ ID NO:120) CTL in No. 8 holes that (i) stimulates respectively illustrates strong IFN-γ respectively compared with the control and generates.These figures Cell of the box indicating on piece on hole in respective aperture is amplified to set up CTL systems.Contrastively, as typical negative number According to MPHOSPH1-A02-9-575 (SEQ ID NO:1) CTL that (j) stimulates does not show that specificity IFN-γ is generated.In figure In, "+" indicator is generated to the IFN-γ for passing through the target cell of suitable peptide impulse, and "-" indicator is to without any peptide impulse The IFN-γ of target cell is generated.
[Fig. 2 a-f] Fig. 2 a-f are made up of one group of line chart (a)-(f), describe the result of IFN-γ ELISA algoscopys, and which shows Show with MPHOSPH1-A02-9-850 (SEQ ID NO:5)(a),MPHOSPH1-A02-9-129(SEQ ID NO:14) (bMPHOSPH1),MPHOSPH1-A02-9-846(SEQ ID NO:64)(c),MPHOSPH1-A02-10-460(SEQ ID NO:73)(d),MPHOSPH1-A02-10-770(SEQ ID NO:77) (e), and MPHOSPH1-A02-10-407 (SEQ ID NO:79) IFN-γ of the CTL systems that (f) stimulates is generated.CTL systems product is measured with IFN-γ enzyme-linked immunosorbent assay (ELISA) The amount of raw IFN-γ.As a result show, the CTL systems for being stimulated with every kind of peptide and being set up all show IFN-γ strong compared with the control Generate.In figure, "+" indicator is generated to the IFN-γ for passing through the target cell of suitable peptide impulse, and "-" indicator is to without any The IFN-γ of the target cell of peptide impulse is generated.R/S ratios show the ratio of responsive cell (CTL systems) and the number for stimulating cell.
[Fig. 2 g-i] Fig. 2 g-i are made up of one group of line chart (g)-(i), describe the result of IFN-γ ELISA algoscopys, and which shows Show with MPHOSPH1-A02-10-923 (SEQ ID NO:97)(g),MPHOSPH1-A02-10-1484(SEQ ID NO:103) (h) and MPHOSPH1-A02-10-282 (SEQ ID NO:120) IFN-γ of the CTL systems that (i) stimulates is generated.Use IFN-γ enzyme Linked immunosorbent assay (ELISA) measures the amount of the IFN-γ of CTL systems generation.As a result show, stimulated with every kind of peptide and set up CTL systems all show strong compared with the control IFN-γ and generate.In figure, "+" indicator is thin to the target for passing through suitable peptide impulse The IFN-γ of born of the same parents is generated, and "-" indicator is generated to the IFN-γ of the target cell without any peptide impulse.R/S ratios show response The ratio of the number of cell (CTL systems) and stimulation cell.
[Fig. 3] Fig. 3 is made up of a series of line chart (a) to (e), is described from MPHOSPH1-A02-9-850 (SEQ ID NO:5)(a),MPHOSPH1-A02-9-846(SEQ IDNO:64)(b),MPHOSPH1-A02-10-460(SEQ ID NO:73) (c),MPHOSPH1-A02-10-770(SEQ ID NO:77) (d) and MPHOSPH1-A02-10-282 (SEQ ID NO:120) The generation of the IFN-γ of the ctl clone that e CTL systems that () stimulates are set up by limiting dilution.As a result demonstrate by with every kind of The ctl clone that peptide stimulates and sets up shows that IFN-γ strong compared with the control is generated.In figure, "+" indicator is to passing through suitable peptide The IFN-γ of the target cell of impulse is generated, and "-" indicator is generated to the IFN-γ of the target cell without any peptide impulse.R/S Than showing the ratio of responsive cell (ctl clone) and the number for stimulating cell.
[Fig. 4] Fig. 4 is the line chart for describing the specific CTL activity for tumor cell line.Using expression MPHOSPH1 and The J82 cells of HLA-A*0201, the HT1376 cells that expresses MPHOSPH1 but do not express HLA-A*0201 and expression HLA-A* 0201 but the T2 cells of MPHOSPH1 are not expressed as stimulating cell.With MPHOSPH1-A02-10-282 (SEQ ID NO:120) The ctl clone of foundation shows the specific CTL activity for J82 cells.On the other hand, it is not detected by being significantly directed to The specific CTL activity of HT1376 and T2 cells.R/S ratios show the ratio of responsive cell (ctl clone) and the number for stimulating cell Example.
[Fig. 5] Fig. 5 is the line chart for describing CTL for the cytotoxic activity of tumor cell line.Using expression MPHOSPH1 and The UMUC-3 cells of HLA-A*0201, the MKN45 cells that expresses MPHOSPH1 but do not express HLA-A*0201 and expression HLA-A* 0201 but the T2 cells of MPHOSPH1 are not expressed as target cell.With MPHOSPH1-A02-10-282 (SEQ ID NO:120) build Vertical ctl clone shows the strong cytotoxic activity for UMUC-3 cells.On the other hand, it is not detected by being significantly directed to The specific CTL activity of MKN45 and T2 cells.E/T ratios show the ratio of effector lymphocyte's (ctl clone) and the number of target cell.
[Fig. 6] Fig. 6 is the line for describing CTL for the cytotoxic activity of the target cell of expression MPHOSPH1 and HLA-A*0206 Figure.The COS7 cells transfected with HLA-A*0206 or total length MPHOSPH1 gene are prepared as control.Use MPHOSPH1-A02- 10-282(SEQ ID NO:120) the CTL systems for setting up are shown for being transfected with both MPHOSPH1 and HLA-A*0206 The specific CTL activity (black diamonds) of COS7 cells.On the other hand, it is not detected by significant for expression HLA-A*0206 The specific CTL activity of the target cell of (triangle) or MPHOSPH1 (circle).
The description of embodiment
Preferred material, device and method is will now be described, but can be made when implementing or check embodiment of the present invention With any method similar or equivalent with method described herein and material and material.However, describe material of the present invention with Before method, it is understood that these descriptions are merely exemplary, it is not intended to limit.It will further be understood that the invention is not restricted to spy Sizing, character, yardstick, material, methodology, scheme etc., because they can change because following routine experiment and/or optimization.This Outward, the term used in the description is simply for description special style or the purpose of embodiment, and not intended to limit this Bright scope, the scope of the present invention only can be limited by claims.
By carrying, state clearly will be complete for the disclosure of each publication, patent or patent application that mentions in this specification Whole it is incorporated herein.However, nowhere may be interpreted as herein recognizing that the present invention is not eligible for by invention formerly and early than these Open.
Unless otherwise defined, all technology used herein and scientific terminology are respectively provided with the technology of relevant art The meaning that personnel generally know.However, if there are conflict, with this specification（Including definition）It is defined.Additionally, these materials, side Method and example are only Illustratives, and are not intended to limit.
I. define
As used in this article, word "/kind ", " being somebody's turn to do " and " described " means " at least one/kind ", unless separately Clearly state.
With material（Such as peptide, antibody, polynucleotide etc.）Associated with term " detached " and " purification " represent the material It is substantially free of at least one other materials potentially included in natural origin.Therefore, the peptide of detached or purification refers to so Peptide, its be substantially free of the cellular material such as carbohydrate, lipid of the cell or tissue that is originated from the peptide or its Its contaminative albumen, or when which is chemosynthesis, it is substantially free of the antibody of precursor or other chemicals.Term " base Cellular material is not contained in sheet " include the peptide that wherein peptide is separated with the cellular component from its cell produced by separation or reorganization Prepared product.Therefore, the peptide for being substantially free of cellular material is included having less than about 30%, 20%, 10% or 5%（By dry weight）Different Source protein matter（Here is also referred to as " contaminating protein matter "）Polypeptide prepared product.When the restructuring of peptide system is produced, which is further preferably substantially Culture medium is not contained, including the peptide prepared product of the culture medium containing less than about 20%, 10% or 5% peptide prepared product volume.When many by changing When learning synthetically produced, its preferably substantially free from precursor or other chemicals, including having less than about 30%, 20%, 10%, 5%（By dry weight）The precursor relevant with peptide symthesis or other chemical drugss peptide prepared product.Particular peptide prepared product contains The peptide of detached or purification can be coagulated by, for example, sodium lauryl sulphate (the SDS)-polyacrylamide in protein prepared product The appearance of single band is showing after coomassie brilliant blue staining of gel electrophoresis and gel etc..In a preferred embodiment In, the peptide and polynucleotide of the present invention are detached or purification.
Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, and refers to the polymer of amino acid residue.The art It can be residue or non-naturally-occurring type residue (the such as phase through modifying that language is applied to wherein one or more amino acid residues The artificial chemical mimetic of the naturally occurring aminoacid that answers) amino acid polymer, and naturally occurring aminoacid polymerization Thing.
Sometimes the term " oligopeptide " used in this specification is used for referring to that length to be 20 residues or less, typically 15 Residue or the peptide of less present invention, generally by about 8-about 11 residues, often 9 or 10 residue compositions.Both afterwards " nonapeptide " and " decapeptide " is referred to as herein.
As used in this article, term " aminoacid " refers to both naturally occurring and synthetic aminoacid, and have with natural The amino acid analogue of the function of the amino acid similarity of presence and amino acid simulant.Aminoacid can be l-amino acid or D- ammonia Base acid.Naturally occurring aminoacid refers to by the aminoacid of genetic code encoding, and adorned ammonia upon translation in cell Base acid（Such as hydroxyproline, Gla and O- phosphoserines）.Phrase " amino acid analogue " refers to be deposited with natural There is identical basic chemical structure in type aminoacid（α carbon is combined with hydrogen, carboxyl, amino and R group）But there is one or many The compound of the individual main chain through the R group of modification or through modification（For example homoserine, nor-leucine, methionine sulfoxide, Methionine methyl sulfonium）.Phrase " amino acid simulant " refer to from have the structure different with general aminoacid but play with general The chemical compound of the function of amino acid similarity.
Aminoacid can pass through three letter symbols known to them or IUPAC-IUB biological chemical name committee members herein The one-letter symbol that can recommend is censuring.
Term " gene ", " polynucleotide " and " nucleic acid " is used interchangeably herein unless expressly stated otherwise, and Censure similarly by their generally accepted single letter codes with aminoacid.
Term " agent " and " compositionss " and it is used interchangeably herein, for referring to the predetermined component comprising ormal weight Product, and any product directly or indirectly obtained by the combination of the predetermined component of the ormal weight.These terms When associating use (such as " medicine agent " and " pharmaceutical composition ") with modifier " medicine ", it is intended that cover：Including active component And the product of the inert fraction of any composition carrier, and the combination by any two or more kinds of compositions, compound Or aggregation, or by the dissociation of one or more component, or the other types of reaction by one or more composition or mutual Any product for acting on and directly or indirectly obtaining.Correspondingly, the present invention linguistic context in, term " medicine agent " and " pharmaceutical composition " refer to any molecule by mixing the present invention or compound and pharmacy or physiologically acceptable carrier and The product that makes.
Phrase " pharmaceutically acceptable carrier " used herein or " physiologically acceptable carrier " means medicine On or physiologically acceptable material, compositionss, material or medium, including but not limited to liquid filler, diluent, Excipient, solvent or embedded material.
The medicine agent or pharmaceutical composition of the present invention especially can serve as vaccine.In the linguistic context of the present invention, phrase " vaccine " (also known as " immunogenic composition) refer to the effect with inducing antitumor immunity function after being inoculated in animal body Agent compositionss.
Term " active component " is here and hereinafter meant that in compositionss the agent with biologic activity or physiologically active Or material.Especially, in the linguistic context of pharmaceutical composition, term " active component " refers to the thing for showing objective pharmacological effect Matter.For example, in the occasion of the pharmaceutical composition for treatment or prophylaxis of cancer, the active component in compositionss can be direct or indirect Ground causes at least one biology or physiological role for cancerous cell and/or tissue.Preferably, such effect may include Reduce or anticancer growth, destruction or kill cancerous cell and/or cancerous tissue, etc..Typically, the indirect effect of active component Fruit includes that induction can recognize that or kill the CTL of cancerous cell.Before preparation, " active component " can claim again " crude drug " (bulk), " drug substance " (drug substance) or " technical products " (technical product).
Unless otherwise defined, term " cancer " refers to the cancer of overexpression MPHOSPH1 genes, the cancer of overexpression MPHOSPH1 The example of disease include but is not limited to bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, Lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.
Unless otherwise defined, term " cytotoxic T lymphocyte ", " cytotoxic T cell " and " CTL " herein may be used Used interchangeably, and unless expressly stated otherwise, refer to recognize non-self cell（Such as lesion/cancer cell, virus infect Cell）And induce the t lymphocyte subset group of such cell death.
Unless otherwise defined, term " HLA-A2 " used herein typically refers to hypotype, and its example includes but do not limit In HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A* 0207,HLA-A*0210,HLA-A*0211,HLA-A*0213,HLA-A*0216,HLA-A*0218,HLA-A*0219,HLA-A* 0228 and HLA-A*0250.
Unless otherwise defined, as used herein, the term " test kit " refers to the combination of reagent and other materials.Consideration Term " test kit " may include microarray, chip, label, etc..Term " test kit " is not intended to be limited to certain and specifically tries Agent and/or combination of materials.
As used in this article, in the linguistic context of experimenter or patient, phrase " the HLA antigens of experimenter (or patient) It is HLA-A2 " have HLA-A2 antigen genes as MHC (Main Tissues phases with referring to experimenter or patient's homozygosis ground or heterozygosis Capacitive complex) I quasi-molecules, and HLA-A2 antigens in the cell of experimenter or patient as HLA antigen presentations.
Useful in the linguistic context of " treatment " cancer with compositionss with the inventive method be limited, if treatment cause clinically Income, such as the size of cancer, universality (prevalence) or the reduction of metastatic potential in subject, cancer progression Postpone, the alleviation of cancer clinical symptom, the prolongation of life span, suppression of recurrence after operation etc., then it is assumed that treatment is " effectively ".When prophylactically application for the treatment of, " effective " means which hinders or prevent the formation of cancer, or prevents or mitigate The clinical symptoms of cancer." effectiveness " is combined and is determined for diagnosing or treating any known method of specific tumors type.
It is limited with the linguistic context that the material of the present invention and method can be used for " prevention " and " strick precaution " cancer, such term is herein In be used interchangeably, refer to reduce because of disease mortality rate or sickness rate bear any activity.Prevention and strick precaution can be betided " one-level, two grades and tertiary prevention level ".Primary prevention and take precautions against and avoid the generation of disease, and the prevention of two grades and three-level level Cover that to be intended to prevent and takes precautions against the progress of disease with the appearance of symptom and related by recovery function and mitigation disease with taking precautions against Complication is reducing the activity of the negative effect of the disease that has set up.Or, prevention and strick precaution may include to be intended to mitigate specific disease The diversified prevention Sex therapy of the seriousness (for example reducing propagation and transfer of tumor etc.) of disease.
In the linguistic context of the present invention, treatment and/or prophylaxis of cancer and/or its recurrence after operation is prevented to include any causing example Behavior such as following events：The induction and cancer that cancerous cell growth suppression, the decline of tumor or regression, cancer go down occurs Prevent, the regression of tumor, the suppression that recurs after the reduction of transfer or suppression, cancer operation, and the prolongation of life span.Cancer Effectively treatment and/or prevention can reduce suffering from cancer individual death rate and improve its prognosis, reduce tumor markerses in its blood Level, and mitigate its detected symptom with cancer.For example, the mitigation of symptom or improvement constitute effectively treatment and/or prevention, Including 10%, 20%, 30% or more reduction, or realize stable disease.
In the linguistic context of the present invention, term " antibody " refers to can there is specific reaction with specified albumen or its peptide Immunoglobulin and its fragment.Antibody can include human antibody, clever long source (primatized) antibody, chimeric antibody, Shuan Te Heterogenetic antibody, humanized antibody and other albumen or the antibody and antibody fragment of radioactive marker fusion.In addition, herein In, antibody is specifically covered intact monoclonal antibodies, polyclonal antibody, is formed by least two complete antibodies with broadest use Multi-specificity antibody（Such as bi-specific antibody）, and antibody fragment, as long as they represent desired biologic activity." anti- Body " indicates all categories（Such as IgA, IgD, IgE, IgG and IgM）.
II. peptide
The peptide of the present invention being detailed below can be described as " MPHOSPH1 peptides " or " MPHOSPH1 polypeptides ".
In order to prove that the peptide from MPHOSPH1 plays the function of the antigen recognized by CTL, to being derived from MPHOSPH1 (SEQ ID NO:126) peptide has carried out analyzing to determine that whether they be the restricted epitopes of HLA-A2, and HLA-A2 is HLA allele (Date Y et al., the Tissue Antigens 47 for frequently encountering:93-101,1996;Kondo A et al.,J Immunol 155:4307-12,1995;Kubo RT et al.,J Immunol 152:3913-24,1994).
Based on the information about their binding affinities to HLA-A2, the HLA-A2 combinations from MPHOSPH1 are identified The candidate of peptide.
Additionally, with after dendritic cell (DC) the stimulated in vitro T cell that these peptide impulses (have been loaded), by using under Stating each peptide stimulates DC to be successfully set up CTL：
MPHOSPH1-A2-9-850(SEQ ID NO:5),
MPHOSPH1-A2-9-129(SEQ ID NO:14),
MPHOSPH1-A2-9-846(SEQ ID NO:64),
MPHOSPH1-A2-10-460(SEQ ID NO:73),
MPHOSPH1-A2-10-770(SEQ ID NO:77),
MPHOSPH1-A2-10-407(SEQ ID NO:79),
MPHOSPH1-A2-10-923(SEQ ID NO:97),
MPHOSPH1-A2-10-1484(SEQ ID NO:103) and
MPHOSPH1-A2-10-282(SEQ ID NO:120)
The CTL of these foundation is directed to and shows strong and specific CTL activity through the target cell of corresponding peptides impulse.These results Prove that MPHOSPH1 is the antigen recognized by CTL, and these are test for the epi-position limited by HLA-A2 that peptide is MPHOSPH1 Peptide；Therefore, these peptides are probably effectively as the target antigen of CTL cytotoxicity.Additionally, MPHOSPH1-A2-10-282 (SEQ ID NO:120) CTL for inducing has the cancerous cell for the expression MPHOSPH1 and HLA-A2 as MHC I quasi-molecules Strong cytotoxic activity.Prompting MPHOSPH1-A2-10-282 is naturally occurring in vivo for the result, and by HLA-A2 antigens (for example HLA-A*0201 or HLA-A*0206) offer on the cancerous cell of expression MPHOSPH1.According to these discoveries, comprising selected from SEQ ID NO:The peptide of 5,14,64,73,77,79,97,103 and 120 aminoacid sequence, or derivatives thereof, mutant, variant or repair Decorations peptide, can be used in the linguistic context of immunization therapy, for treating the cancer of expression MPHOSPH1 and HLA-A2 antigens.At this In bright particular, the peptide being made up of aminoacid sequence disclosed herein can be used for the immunization therapy of cancer.Control The cancer for the treatment of include but is not limited to bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, Lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.But, the peptide of the present invention can be applicable to any cancer, as long as They express MPHOSPH1 and HLA-A2 antigens.
As MPHOSPH1 genes are in cancerous cell, such as bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, knot are straight Overexpression in intestinal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms, and in great majority just Often do not express in organ, it is good immunotherapeutic targets.Therefore, the present invention provides being known by CTL from MPHOSPH1 The nonapeptide of other epi-position（The peptide being made up of nine amino acid residues）And decapeptide（The peptide being made up of ten amino acid residues）.Or Person, the present invention provide the detached peptide for combining HLA antigens and inducing cytotoxic T lymphocytes (CTL), wherein described peptide by MPHOSPH1(SEQ ID NO:126) immunologic competence fragment is constituted.More specifically, the present invention is provided comprising selected from SEQ ID NO:The peptide of 5,14,64,73,77,79,97,103 and 120 aminoacid sequence.More specifically, in some embodiments, this The peptide of invention is comprising SEQ ID NO:The peptide of 120 aminoacid sequence.
In general, the software program of such as internet access, such as Parker KC et al., J can be passed through at present Immunol 1994,Jan 1 152(1):163-75, and Nielsen M et al., Protein Sci 2003;12:1007- Those described in 17 grades, can be used to calculate the binding affinity between different peptides and HLA antigens on computers.With HLA The binding affinity of antigen can be according to being measured described in such as following documents：Parker KC et al.,J Immunol 1994,Jan 1 152(1):163-75, Kuzushima K et al., Blood 2001,98 (6):1872-81, Larsen MV et al.BMC Bioinformatics.2007 Oct 31;8:424,Buus S et al.Tissue Antigens.,62:378-84,2003,Nielsen M et al.,Protein Sci 2003;12:1007-17, and Nielsen M et al.PLoS ONE 2007;2:E796, its are summarized in such as Lafuente EM et al., Current Pharmaceutical Design,2009,15,3209-3220.For determining the method for binding affinity in such as Journal of Immunological Methods,(1995,185:181-190);With Protein Science (2000,9:1838- 1846) it is described in.Thus the HLA antigen bindings with height can be selected affine easily with such software program The fragment from MPHOSPH1 of power.Therefore, the present invention is covered and will be confirmed as being resisted with HLA by these known procedures by any Former peptide combining, constituting derived from MPHOSPH1 fragments.Additionally, such peptide is may include by the full length sequence group of MPHOSPH1 Into peptide.
The peptide of the present invention, especially nonapeptide of the invention and decapeptide, can have extra amino acid residue in its flank, As long as these peptides keep their CTL inducibilities.Specific additional amino acid residue can be by any kind of amino Acid is constituted, as long as which does not damage the CTL inducibilities of original peptide.Therefore, the present invention covers the peptide with CTL inducibilities, Wherein described peptide includes the aminoacid sequence derived from MPHOSPH1.Such peptide is such as less than about 40 aminoacid, often Less than about 20 aminoacid, generally less than about 15 aminoacid.
One in known peptide, two, the modification of several or multiple aminoacid do not affect the function of peptide, or in some situations The lower desired function that can even strengthen former peptide.In fact, it is known to through the peptide of modification（I.e. by compared with original canonical sequence its Middle modification（I.e. by by the replacement of former reference sequences, insertion, disappearance and/or interpolation one, two or several amino acid residues The peptide that the aminoacid sequence of modification is constituted）Retain biologic activity (Mark et al., the Proc Natl Acad of original peptide Sci USA 1984,81:5662-6;Zoller and Smith,Nucleic Acids Res 1982,10:6487-500; Dalbadie-McFarland et al.,Proc Natl Acad Sci USA 1982,79:6409-13).Therefore, at this In a bright embodiment, the peptide with CTL inducibilities of the present invention can be made up of such peptide, and the peptide has choosing From SEQ ID NO:5,14,64,73,77,79,97,103 and 120 aminoacid sequence, wherein add, disappearance, insertion, and/or Instead of one, two or several aminoacid.
In another embodiment, peptide of the invention can be such peptide, and which is included in selected from SEQ ID NO:5, Replace in 14,64,73,77,79,97,103 and 120 aminoacid sequence, lack, inserting and/or adding one, two or several The aminoacid sequence of individual aminoacid, condition are the CTL inducibilities that the modified peptides retain original peptide.In preferred embodiment In, the peptide of the present invention can be included in SEQ ID NO:Replace in 120 aminoacid sequence, lack, inserting and/or adding one The peptide of the aminoacid sequence of individual, two or several aminoacid, condition are the CTL inducibilities that the modified peptides retain original peptide.
It will be appreciated by those skilled in the art that changing single amino acids or minority percentage ratio ammonia in overall amino acid sequence Indivedual modifications (add, insert, lack or replace) of base acid often lead to the characteristic of Original amino side chain and are retained； Therefore which is referred to as " conservative replacement " or " conservative modification ", wherein causes the albumen with similar function to the change of protein Matter.The conservative replacement table for providing functionally similar aminoacid is well known in the art.The example of amino acid side chain feature includes Such as hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T) and tool There is the side chain of following common functional group or feature：Aliphatic lateral chain (G, A, V, L, I, P)；Hydroxyl side chain (S, T, Y)；Sulfur-bearing Atom side chain (C, M)；Containing carboxylic acid and amide side chains (D, N, E, Q)；Side chain containing alkali (R, K, H)；With containing beta-branched side (H, F, Y, W).In addition, the following eight groups aminoacid each containing conservative replacement each other：
1) alanine (A), glycine (G)；
2) aspartic acid (D), glutamic acid (E)；
3) agedoite (N), glutamine (Q)；
4) arginine (R), lysine (K)；
5) isoleucine (I), Leucine (L), methionine (M), L-Valine (V)；
6) phenylalanine (F), L-Tyrosine (Y), tryptophan (W)；
7) serine (S), threonine (T)；With
8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins is 1984).
Such conservative modified peptides are also regarded as the peptide of the present invention.However, the peptide not limited to this of the present invention, it may include non-conservative Modification, as long as the peptide retains the CTL inducibilities of original unmodified peptide.In addition, through modification peptide should not exclude derived from The peptide of the inducible CTL in the polymorphie variant of MPHOSPH1, inter-species congener or allele.
To the insertion of the peptide of the present invention, replacement, disappearance and/or amino acid residue can be added, or, can be from the present invention's Peptide deleted amino acid residues, to realize higher binding affinity.For the CTL inducibilities needed for keeping, can modify and (insert Enter, lack, add and/or replace) aminoacid of minority (for example, 1,2 is several) or little percentage ratio.Here, term is " several Individual " mean the aminoacid of less than 5, such as 4 or less than 3.The percentage ratio of adorned aminoacid is wanted to be preferably less than 20%, Such as less than 15%, more preferably less than 10%, even more preferably or 1-5%.
When used in the linguistic context of immunotherapy for cancer, the peptide of the present invention as with the complex of HLA antigens can be in Pass on the surface of cell or allochthon.It is therefore preferable that selecting not only to can induce CTL but also be also equipped with to HLA antigens The peptide of high binding affinity.For this purpose it is proposed, can be modified to peptide by replacing, inserting, lack and/or add amino acid residue To produce the modified peptides that binding affinity improves.In addition to the natural peptide being demonstrated, since it is known that by anti-with reference to HLA Former and sequence rule (the J Immunol 1994,152 of peptide that are demonstrated:3913;Immunogenetics1995,41:178;J Immunol 1994,155:4307), the immunogenic peptide of the present invention can will be introduced based on the modification of such rule.
For example, show that second aminoacid of N-terminal of the peptide of high HLA-A2 binding affinities tends to be substituted by bright ammonia Acid or methionine.Similarly, C-terminal aminoacid is substituted by L-Valine or leucic peptide can also be preferably used.Therefore, Have and be selected from SEQ ID NO:5,14,64,73,77,79,97,103 and 120 aminoacid sequence, wherein described SEQ ID NO Second aminoacid of N-terminal of aminoacid sequence be substituted by Leucine or methionine, and/or wherein described SEQ ID NO The C-terminal of aminoacid sequence be substituted by L-Valine or leucic peptide is covered within the present invention.In another embodiment In, the present invention covers the peptide with such aminoacid sequence, is selected from SEQ ID NO in the aminoacid sequence:5,14,64, Second aminoacid of N-terminal of 73,77,79,97,103 and 120 aminoacid sequence is substituted by Leucine or methionine, and/ Or the C-terminal of the aminoacid sequence of the SEQ ID NO is substituted by L-Valine or Leucine.In preferred embodiments, originally The peptide of invention can include such aminoacid sequence, wherein SEQ ID NO:Second aminoacid of N-terminal of 120 aminoacid sequence Be substituted by Leucine or methionine, and/or the C-terminal of the aminoacid sequence of the SEQ ID NO be substituted by L-Valine or Leucine.
In one embodiment, the present invention provide have CTL inducibilities peptide, wherein described peptide have be selected from the group (1) to the formula of (9)：
(1)-correspond to MPHOSPH1-A2-9-850 (SEQ ID NO:5)-
F[X1]L T I E N E[X2],
(2)-correspond to MPHOSPH1-A2-9-129 (SEQ ID NO:14)-
F[X1]G C I M Q P[X2],
(3)-correspond to MPHOSPH1-A2-9-846 (SEQ ID NO:64)-
G[X1]R A F L L T[X2],
(4)-correspond to MPHOSPH1-A2-10-460 (SEQ ID NO:73)-
Y[X1]A Y D E T L N[X2],
(5)-correspond to MPHOSPH1-A2-10-770 (SEQ ID NO:77)-
K[X1]I C N E T V E[X2]
(6)-correspond to MPHOSPH1-A2-10-407 (SEQ ID NO:79)-
L[X1]T L G K C I N[X2]
(7)-correspond to MPHOSPH1-A2-10-923 (SEQ ID NO:97)-
K[X1]S N E I E T A[X2]
(8)-correspond to MPHOSPH1-A2-10-1484 (SEQ ID NO:103)-
Q [X1] V A A L E I Q [X2], and
(9)-correspond to MPHOSPH1-A2-10-282 (SEQ ID NO:120)-
Y[X1]Y D L F V P V[X2].
In formula (1)-(9), [X1] is Leucine or methionine, and [X2] is L-Valine or Leucine.At this In a bright particularly preferred embodiment, formula can be (9), and which corresponds to SEQ ID NO:120.The present invention also provide by The detached peptide that formula (1)-(9) above represent, wherein with the addition of one, two or several amino in its N-terminal and/or C-terminal Acid.In an alternative embodiment, the present invention provides the detached peptide represented by formula (1)-(9) above, wherein in its N End and/or C-terminal have lacked one, two or several aminoacid.The present invention was also provided by dividing that formula (1)-(9) above represent From peptide, Anywhere insertion wherein in the sequence has lacked one, two or several aminoacid.
Not only can introduce at the end amino acid of peptide and replace, and can know in the potential φt cell receptor (TCR) of peptide Introduce at other position and replace.Several study the peptide that has been proved with aminoacid replacement can be equal to or be better than original, for example CAP1、p53(264-272)、Her-2/neu(369-377)Or gp100(209-217)(Zaremba et al.Cancer Res.57,4570- 4577,1997,T.K.Hoffmann et al.J Immunol.(2002);168(3):1338-47.,S.O.Dionne et al.Cancer Immunol immunother.(2003)52:199-206 and S.O.Dionne et al.Cancer Immunology,Immunotherapy(2004)53,307-314).
Present invention further contemplates that the N and/or C-terminal interpolation one, two or several aminoacid to the peptide of the present invention.Such reservation The modified peptides of CTL inducibilities are also contained within the present invention.
However, when peptide sequence is a part of identical with the aminoacid sequence of the endogenous or exogenous proteins with difference in functionality When, side effect, such as autoimmune conditions or the allergic symptoms for predetermined substance may be induced.Therefore, it can utilize Available data base implements homology search, with the feelings for avoiding the sequence of peptide from mating with the aminoacid sequence of another kind of protein Condition.When having understood that even if the peptide for only having 1 or 2 amino acid of differences to target peptide is not also present according to homology search, can Its binding affinity with HLA antigens is improved to modify target peptide, and/or improves its CTL inducibility, without any The danger of the such side effect of life.
Although there is the peptide of high binding affinity to be contemplated to be highly effective HLA antigens as above, but right The presence that the candidate peptide that index selects checked CTL inducibilities existed for according to high binding affinity.Here, phrase " CTL Inducibility " refers to peptide induces CTL ability when being presented to antigen-presenting cell (APC) above.In addition, " CTL inducibilities " bag Inducing peptide CTL activation, CTL propagation is included, is promoted CTL dissolving target cells and is improved the ability that CTL IFN-γs are generated.
CTL inducibilities are identified through such as realization of getting off, and (such as B- lymphs are thin to induce the APC of carrier's MHC antigens Born of the same parents, macrophage and dendritic cell (DC)), or more specifically, from the DC of human peripheral monocyte, and Stimulated after APC with test peptides, APC is mixed with CD8- positive cells to induce CTL, then measured and generated and discharged by CTL IFN-γ for target cell.As response system, can be using the transgenic animal (example of the expression people's HLA antigens that has made Such as BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000,Aug,61(8):764-79Related Articles,Books,Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1transgenic mice: Those described in dependent on MHC (HLA) class II restricted T (H) response).Or, can be with With51The radioactive label target cell such as Cr, and the cellular cytoxicity activity of CTL can be calculated from the radioactivity of target cell release.Or Person, it can assess as follows：The IFN-γ in the presence of the cell for carrying immobilization peptide being generated and discharged by CTL is measured, and is made Manifest the inhibition zone in culture medium with anti-IFN-γ monoclonal antibody.
As the result for checking the CTL inducibilities of peptide as mentioned above, find selected from selected from SEQ ID NO:5,14, It is affine that not only there is the nonapeptide or decapeptide of 64,73,77,79,97,103 and 120 aminoacid sequence the height to HLA antigens to combine Power, also has CTL inducibilities.Therefore, these peptides are enumerated for the preferred embodiment of the invention.
In addition, homology analysis result shows these peptides and does not have from peptide derived from any other known people's gene product There is significant homology.There is the probability of unknown or undesired immunne response when it reducing for immunotherapy.Cause This, and according in terms of this, these peptides can be used to cause the immunity for MPHOSPH1 in cancer patient.Therefore, originally Invention is covered with selected from SEQ ID NO:The peptide of 5,14,64,73,77,79,97,103 and 120 aminoacid sequence.
In addition to modification discussed above, the peptide of the present invention can be connected to other peptides, as long as the connection peptides of gained retain The CTL inducibilities of original peptide.The example of suitably " other peptides " includes：The present invention peptide or from energy derived from other TAA The peptide of induction CTL.The peptide of the present invention can connect " other " peptide indirectly directly or through joint.Joint between peptide is this Known to field, such as AAY (P.M.Daftarian et al., J Trans Med 2007,5:26),AAA,NKRK (R.P.M.Sutmuller et al.,J Immunol.2000,165:7308-7315) or K (S.Ota et al., Can Res.62,1471-1476,K.S.Kawamura et al.,J Immunol.2002,168:5709-5715).
For example, the peptide of the tumor associated antigen derived from non-MPHOSPH1 may also be used for increasing by HLA I classes and/or The immunne response of II classes.Known cancerous cell expression exceedes a kind of tumor-related gene.Some CTL derived from such TAA can Induction peptide has been separated (such as WO2008/047473, WO2010/047062, WO2008/102557, WO2009/ 025116).Therefore, be connected to the present invention peptide " other " peptide example include but is not limited to derived from MPHOSPH1 outside The inducible peptides of the CTL of TAA.In the present invention, " other " peptide can be not only MHC I class restricted type peptides, and page can be MHC II Class restricted type peptide.Those skilled in the art can be prepared including one or more using conventional molecular biology program MPHOSPH1 peptides and the polypeptide of one or more non-MPHOSPH1 peptide, or the nucleic acid of encoding such polypeptides.
Above-mentioned connection peptides are referred to herein as " multi-epitope " (polytope) ", i.e. two or more potential immunity The group that originality or immunne response stimulatory peptides are constituted, these peptides can be with various arrangement modes（For example connect, overlap）It is connected to Together.The multi-epitope（Or encode the nucleic acid of the multi-epitope）Can apply according to standard immunization protocol, for example, be applied to animal, with Testing the multi-epitope stimulates, strengthens and/or cause the effectiveness of immunne response.
Peptide can be directly or through being joined together to form multi-epitope using flanking sequence, and multi-epitope is used as vaccine Purposes is well known in the art (to see, for example, Thomson et al., Proc.Natl.Acad.Sci USA 92 (13):5845- 5849,1995;Gilbert et al.,Nature Biotechnol.15(12):1280-1284,1997;Thomson et al.,J Immunol.157(2):822-826,1996;Tarn et al.,J Exp.Med.171(l):299-306,1990). The multi-epitope containing different number and combination of epi-positions can be prepared and test situation and the raising immunne response that they are recognized by CTL Effect.
The peptide of the present invention can be further attached to other materials, as long as they retain CTL inducibilities.Such " other " The illustrative example of material is included but is not limited to：Peptide, lipid, sugar and sugar chain, acetyl group, the polymer of natural and synthesis etc.. Peptide can also contain modification, such as glycosylation, oxide side chain or phosphorylation；On condition that the modification do not destroy described herein such as peptide Biologic activity.The modification of these types can be carried out to give extra function（Such as target function and delivery function）Or Make stabilizing polypeptides.
For example, in order to improve the internal stability of polypeptide, introducing D- aminoacid known in the art, amino acid simulant or non- Natural amino acid；This design is equally applicable to polypeptide of the present invention.The stability of polypeptide is determined in many ways can.For example, may be used Using peptidase and various biological medias（Such as human plasma and serum）Carry out measuring stability and (see, for example, Verhoef et al.,Eur J Drug Metab Pharmacokin1986,11:291-302).
When the peptide of the present invention includes cysteine residues, peptide is easy between the SH groups by cysteine residues two Sulfide linkage forms dimer.Therefore, peptide of the invention also includes the dimer of the peptide of the present invention.
Additionally, as described above, replacing, lacking, inserting, and/or with the addition of one, two or several amino acid residues Modified peptides in, can screen or select and the same or higher person of original peptide phase specific activity.Therefore, the present invention is also provided due to sieve Choosing or the method for selecting the modified peptides same or higher with original peptide phase specific activity.For example, methods described may include following step：
a:Peptide modification (replace, lack, insert or add) at least one amino acid residue to the present invention,
b:In determination step (a), modification obtains the activity of peptide, and
c：Select the peptide with same or higher activity compared with original peptide.
Here, the activity may include MHC binding activity and APC or CTL inducibilities.Preferably, the activity of peptide is CTL Inducibility.
The preparation of III.MPHOSPH1 peptides
The peptide of the present invention can be prepared using known technology.For example, peptide can pass through synthesis, using recombinant DNA technology or Chemosynthesis are preparing.The peptide of the present invention individually can synthesize, or synthesize the longer polypeptide including two or more peptides.So After can separate, i.e. purification or the separation peptide so as to be substantially free of other naturally occurring host cell proteins matter and its Fragment or any other chemical substance.
The peptide of the present invention can contain modification, glycosylation, oxide side chain or phosphorylation etc., on condition that the modification is not broken The biologic activity of bad original peptide.The modification of other examples includes mixing D- aminoacid or other available amino acid analogs Thing, for example to increase the serum halflife of peptide.
According to selected aminoacid sequence, the peptide of the present invention can be obtained by chemosynthesis.It is applicable to synthesis The example of conventional peptide synthesis includes：
(i)Peptide Synthesis,Interscience,New York,1966；
(ii)The Proteins,Vol.2,Academic Press,New York,1976；
(iii) Peptide Synthesis (Japanese), Maruzen Co., 1975；
(iv) Basics and Experiment of Peptide Synthesis (Japanese), Maruzen Co., 1985；
(v) Development of Pharmaceuticals (second volume) (Japanese), Vol.14 (peptide synthesis),Hirokawa,1991；
(vi)WO99/67288；With
(vii)Barany G.&Merrifield R.B.,Peptides Vol.2,″Solid Phase Peptide Synthesis″,Academic Press,New York,1980,100-118.
Or, peptide (the such as Morrison of the present invention can be obtained using any of genetic engineering peptide production method J,J Bacteriology1977,132:349-51;Clark-Curtiss&Curtiss,Methodsin Enzymology (eds.Wu et al.)1983,101:347-62).For example, first, prepare comprising in can expression-form（For instance in suitable Regulatory sequence downstream in promoter sequence）Encoding target peptide polynucleotide suitable carrier, and be transformed into suitable place Chief cell.The present invention also provides such carrier and host cell.Then cultivate host cell to generate peptide interested.Also may be used To produce peptide in vitro using external translating system.
IV. polynucleotide：
The present invention also provides the polynucleotide of any of above peptide of the present invention of coding.The polynucleotide of the present invention may include to be derived from Naturally occurring type MPHOSPH1 gene (for example, GenBank Accession No.NM_001031702 (SEQ ID NO:125)) Polynucleotide and through with them guarding modification nucleotide sequence polynucleotide.Herein, phrase " is passed through The nucleotide sequence of conservative modification " refers to identical or substantially identical aminoacid sequence the sequence of coding.Due to genetic code Degeneracy, has on extremely several functions identical nucleic acid for any given protein to encode it.For example, codon GCA, GCC, GCG and GCU all coded amino acid alanine.Therefore, in any position for being defined as alanine by codon, this is close Numeral can change over described any corresponding codon, and not change coded polypeptide.Such variance is in this area It is referred to as " silent variant ", represents one kind of conservative modification variant.Each is described as the nucleic acid sequence of encoded peptide herein Row also describe each possible silent variant of the nucleic acid.Those skilled in the art are it is readily appreciated that each in nucleic acid Individual codon（Except AUG and TGG, AUG is the unique codon of methionine under normal circumstances, and TGG is under normal circumstances It is the unique codon of tryptophan）Can be modified to produce functionally identical molecule.Thus, each coding disclosed The nucleotide sequence of peptide represents the implicit disclosure of associated silent variant.
The polynucleotide of the present invention can be made up of DNA, RNA and its derivant.As it is known in the art, DNA molecular by Such as naturally occurring base A, T of base, C and G are suitably constituted, and T is replaced by U in RNA.Those skilled in the art's meeting Recognize that non-naturally occurring base is also included within polynucleotide.
The peptide of the multiple present invention of polynucleotide codified of the invention, is wherein present with or without aminoacid sequence between two parties.Example Such as, aminoacid sequence can provide the cleavage site of peptides that are polynucleotide or being translated between two parties（Such as enzyme recognition sequence）.In addition, The polynucleotide of the present invention may also include any extra sequence in addition to the coded sequence of peptide of the present invention is encoded.For example, this Bright polynucleotide can be the recombination of polynucleotide for including the regulatory sequence required for expression of peptides, or can be with mark The expression vector of gene etc.（Plasmid）.In general, such recombination of polynucleotide can operate multinuclear by conventional recombinant techniques Thuja acid preparing, such as by using polymerase and endonuclease.
Restructuring and chemical synthesising technology can be adopted to the polynucleotide for generating the present invention.For example, it is possible to pass through will with this The polynucleotide of the coded sequence of invention peptide are inserted in the appropriate carrier that can be expressed after competent cell is transfected into generate The polynucleotide of the present invention.Or, the multinuclear of the present invention can be expanded by round pcr or by the duplication in suitable host Thuja acid (see, for example, Sambrook et al., Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York,1989).Or, the multinuclear of the present invention can be synthesized using solid phase technique Thuja acid, such as Beaucage SL＆Iyer RP, Tetrahedron 1992,48:2223-311;Matthes et al.,EMBO J 1984,3:Described in 801-5.
V. allochthon (exosomes)：
Invention further provides the intracellular vesicles of referred to as allochthon, these allochthons are presented over their surface There is the complex formed between peptide of the present invention and HLA antigens.Allochthon can be by, for example, in Japanese patent application public affairs table publication The method described in detail in flat 11-510507 and WO99/03499 is preparing, it is possible to from as treatment and/or object of prevention Patient obtain APC prepare.The allochthon of the present invention can be connect in the way of similar to the peptide of the present invention as vaccine Kind.
The type of the HLA antigens included in complex must be with needs treatment and/or the type matching of the experimenter of prevention. For example, for Japanese, HLA-A2, especially HLA-A*0201 and HLA-A*0206 are probably usually suitable.Use The HLA-A2 types that expresses in Japanese and white people's camber are favourable for effective result is obtained, and can use The hypotypes such as HLA-A*0201 and HLA-A*0206.Typically, clinically, the HLA antigens of patient in need for the treatment of are investigated in advance Type, this makes it possible to suitably selected this antigen is had high-caliber binding affinity or have by antigen be in The peptide of the CTL inducibilities that passs.Additionally, in order to obtain the peptide for showing high binding affinity and CTL inducibilities, can be natural Carry out on the basis of the aminoacid sequence of the MPHOSPH1 partial peptides of presence the replacement of 1,2 or several aminoacid, disappearance or Add.
When the allochthon for the present invention uses HLA-A2 type HLA antigens, with SEQ ID NO:5,14,64,73,77, The peptide of the arbitrary aminoacid sequence in 79,97,103 and 120 is useful especially.In certain embodiments, the present invention's is outer It is to present the allochthon for having the peptide with the complex of HLA-A2 antigens of the present invention in its surface to carry out body.Include in such complex HLA-A2 antigens exemplary include but is not limited to HLA-A*0201 and HLA-A*0206.
VI. antigen-presenting cell (APC)：
The present invention be additionally provided on its surface present HLA antigens with the present invention peptide formed complex detached APC.The APC may originate from the patient as treatment and/or object of prevention, and can be as vaccine individually or and other medicines （Peptide, allochthon or CTL including the present invention）Combine to apply.
APC is not limited to particular kind of cell, and including dendritic cell (DC), Langerhans cells, macrophage, B cell and activation T cell, it is known that these cells can on their cell surface presenter protein property antigen, for Lymphocyte is recognized.As DC is the representative APC in APC with most strong CTL induced activitys, DC is suitable as of the invention APC.
For example, can pass through to induce DC from peripheral blood lymphocytes, then in vitro or in vivo with the peptide of the present invention Contact（Stimulate）They are obtaining APC.When the peptide of the present invention is applied to experimenter, induce presentation in the body of experimenter The APC of peptide of the present invention.Therefore, it can using the peptide of the present invention, then collect APC from experimenter, obtain by experimenter The APC of the present invention.Or, can obtain the present invention's by making the APC collected from experimenter contact with the peptide of the present invention APC.
The present invention APC can individually or and other medicines, including the present invention peptide, allochthon or CTL combined administration Experimenter is given, so that the immunne response of cancer is directed in experimenter's Immune inducing in vivo.For example, in vitro administration may include following step：
a：APC is collected from the first experimenter；
b：Make the APC of peptide contact procedure a of the present invention；And
c：APC to second experimenter's step b.
First experimenter and the second experimenter can be same individualities, or can be Different Individuals.Step b is obtained APC can be treated as vaccine administration and/or prophylaxis of cancer, and the example of cancer includes but is not limited to bladder cancer, breast carcinoma, palace Neck cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue swell Tumor.
The present invention also provides method of the manufacture for inducing the pharmaceutical composition of APC, and wherein methods described is included this The step of bright peptide is mixed with pharmaceutically acceptable carrier or is prepared.
According to one aspect of the present invention, the APC of the present invention has CTL inducibilities.In the linguistic context of APC, phrase " tool Have CTL inducibilities " refer to and the higher CTL inducibilities of display are not compared with the APC of peptide contact.Such lure with CTL The APC of ability is led in addition to the method outlined supra, is additionally included in external by containing the polynucleotide for encoding peptide of the present invention Gene transfer is prepared to method the step of APC.The gene for being imported can be the form of DNA or RNA.For being imported The example of method include but be not specifically limited to the various methods that routinely implements in this area, such as can be turned using liposome Dye, electroporation or calcium phosphate method.In particular, can be such as Cancer Res 1996,56:5672-7;J Immunol 1998,161:5607-13;J Exp Med 1996,184:465-72;International Publication text No.2000-509281's is disclosed Implement described in translator of Japanese.By the gene transfer entered APC, gene experience in cell transcription, translation, etc., Then the protein for obtaining is processed by MHC I classes or II classes, and via presentation approach presenting partial peptide.
In some embodiments, APC of the invention is to present HLA-A2 antigens over their surface with the present invention The APC of the complex of peptide.The representative instance of the HLA-A2 antigens included in such complex includes but is not limited to HLA-A*0201 And HLA-A*0206.
VII. cytotoxic T cell (CTL)：
The CTL for any peptide of the present invention for inducing can strengthen the immunne response of target cancer cell in vivo, therefore may be used By being used as vaccine in the way of similar to the peptide.Therefore, the present invention is provided by any peptide specific induction of the present invention or is activated , detached CTL.
Such CTL can be obtained by following step：(1) peptide of the present invention is applied to experimenter；(2) in vitro with this Bright peptide contact（Stimulate）It is derived from APC the and CD8- positive T cells or peripheral blood mononuclear leukocyte of experimenter；Or (3) are in body CD8- positive T cells or the contact of peripheral blood mononuclear leukocyte is made outward to present in its surface and have shape between HLA antigens and the peptide Into complex APC or allochthon；Or (4) import polynucleotide of two φt cell receptor (TCR) subunits of coding, Or encode multiple polynucleotide of each TCR subunit, wherein described TCR can in conjunction with the HLA-A2 antigens on cell surface with The complex of the peptide of the present invention.Such APC or allochthon that will be used in prepared by CTL can be made by the method being described above Standby.(4) details of method are below described in " VIII.T cell receptors (TCR) " part.
The CTL of the present invention can be obtained from the patient that will be treated and/or prevented, and can individually be applied them With, or the drug regimen for including the peptide, APC or allochthon of the present invention with other applied producing regulating effect.Obtained CTL specificitys work for the target cell of the peptide (for example with the peptide identical peptide for induction) for presenting the present invention.Target cell Can be the cell of endogenous expression MPHOSPH1, such as cancerous cell, or or the cell that transfected by MPHOSPH1 genes；And because The stimulation of peptide of the present invention and on cell surface present have peptide of the present invention cell also act as activation CTL attack target.
In certain embodiments, CTL of the invention identifications presents the complex for having HLA-A2 antigens and the peptide of the present invention Cell.In the linguistic context of the CTL, phrase " identification cell " refer to by its TCR combine cell surface on HLA-A2 antigens with The complex of the peptide of the present invention, and show the specific cell cytotoxic activity for the cell.Herein, " specific cytotoxic is lived Property " refer to for the cell for presenting HLA-A2 antigens and the complex of the peptide of the present invention, and be not for other cells and show cells Cytotoxic activity.The representative instance of the HLA-A2 antigens included in such complex includes but is not limited to HLA-A*0201 and HLA-A* 0206.
VIII.T cell receptors (TCR)：
The present invention also provides compositionss, and which includes polynucleotide of two TCR subunits of coding, or encodes each Multiple polynucleotide of TCR subunits, wherein described TCR can be in conjunction with the HLA-A2 antigens on cell surface and peptides of the invention Complex, and the method using said composition.There is such TCR subunits the ability for forming such TCR, the TCR to assign T cell is given with the specificity of the tumor cell for expression MPHOSPH1.By using methods known in the art, it is possible to identify go out It is separately encoded the polynucleotide (WO2007/032255 and Morgan of the α and β chains of the TCR of the CTL gone out with the inducing peptide of the present invention et al.,J Immunol,171,3288(2003)).For instance, it may be preferable to use PCR method.PCR primer for analyzing can be with It is, for example, as 5 '-R primers (5 '-gtctaccaggcattcgcttcat-3 ') (SEQ ID NO of 5 ' side primers:127), And the 3-TRa-C primer (5 '-tcagctggaccacagccgcagcgt- special to TCR α chain C areas as 3 ' side primers 3′)(SEQ ID NO:128), the 3-TRb-C1 primer (5 '-tcagaaatcctttctcttgac- special to TCR β chain C1 areas 3′)(SEQ ID NO:129), or special to TCR β chain C2 areas 3-TRbeta-C2 primers (5 '- ctagcctctggaatcctttctctt-3′)(SEQ ID NO:, but not limited to this 130).Derivative TCR can be with high affinity Power is combined and presents the target cell for having MPHOSPH1 peptides, and is optionally directed to and presents the present invention's with mediation in vitro in vivo The Efficient killing effect of the target cell of MPHOSPH1 peptides.
Can will encode polynucleotide of two TCR subunits or encode multiple many nucleoside of each TCR subunit Acid mixes suitable carrier, such as in retroviral vector.These carriers are well known in the art.Can be by many nucleoside Acid or the carrier comprising them usefully proceed to T cell (for example, CD8 positive T cells), such as in the T cell of patient. Advantageously, the present invention provides one kind i.e. with instant compositionss, and which allows the Rapid Modification patient T cell of oneself（Or other The T cell of mammal）Rapidly and easily to generate the modification type T cell for killing characteristic with remarkable cancerous cell.
Specific TCR for the peptide of the present invention should be able to specifically recognize the peptide and HLA antigens of the present invention Complex, gives T cell compound with HLA antigens for the peptide for presenting the present invention when the TCR is presented on the surface of T cell The specificity of the target cell of thing.Activity required for being confirmed by any known method, by importing the such TCR of coding The polypeptide of subunit and the CTL for preparing being capable of the such target cell of specific recognition.The preferred example of such method includes, for example, Determined using the staining analysiss of HLA polymers and ELISPOT of HLA molecules and the peptide of the present invention.Surveyed by implementing ELISPOT Fixed, can confirm that the CTL that prepared by said method can specificity TCR identification cell, and the letter produced by the recognition reaction Number can transmit in the cell.In addition it is also possible to the CTL for being identified through above-mentioned method preparation by known method has pin Specific cell cytotoxic activity to target cell.The example of such method includes, for example, using expression HLA-A2 antigens with The Cr releases of both MPHOSPH1 are determined.
In one aspect, the present invention provides through every with the polynucleotide or coding of two TCR subunit polypeptides of coding The multiple polynucleotide transduction of one TCR subunit and the CTL for preparing, wherein described TCR can in conjunction with cell surface by Have and be selected from SEQ ID NO:The MPHOSPH1 peptides and HLA-A2 of 5,14,64,73,77,79,97,103 and 120 aminoacid sequences The complex of antigen.
CTL through transduceing can go back to the nest (homing) in vivo to cancerous cell, and be trained known to can utilizing in vitro Foster method expands (such as Kawakami et al., J Immunol., 142,3452-3461 (1989)).The present invention can be utilized T cell forming immunogenic composition, the compositionss can be used in the patient for needing treatment or protection treatment and/ Or prophylaxis of cancer (WO2006/031221).
IX. pharmaceutical composition：
Due to MPHOSPH1 expression, in cancer, (example of cancer includes but is not limited to bladder cancer, breast carcinoma, cervix uteri Cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue swell Tumor) in compared with normal structure raise, the present invention peptide or polynucleotide can be used for treatment and/or prophylaxis of cancer.Therefore, this Bright offer is formulated as medicine agent or compositionss for treatment and/or prophylaxis of cancer, and the agent or compositionss include The peptide or polynucleotide of one or more present invention is used as active component.Or, any of above presentation has peptide of the present invention and HLA The allochthon or APC of the complex of antigen can serve as the active component of medicine agent or compositionss.In addition, above-mentioned recognizable The CTL for presenting the cell of the peptide and HLA antigens that have the present invention also is used as the work of medicine agent of the present invention or pharmaceutical composition Property composition.
Therefore, the present invention provides agent or compositionss, and which includes selected from following at least one active component：
The peptide of (a) one or more present invention；
One or more of (b) in can expression-form coding peptide as disclosed herein polynucleotide；
The APC or allochthon of (c) one or more present invention；With
The CTL of (d) one or more present invention.
The medicine agent or compositionss of the present invention also acts as vaccine.In the linguistic context of the present invention, phrase " vaccine "（? Referred to as " immunogenic composition "）Refer to the function with improvement, increase and/or inducing antitumor immunity power after animal is inoculated with Compositionss.In other words, the invention provides suitable for induction among experimenters for the present invention's of the immunne response of cancer Medicine agent or compositionss.
The medicine agent or compositionss of the present invention can be used for treatment and/or prophylaxis of cancer in experimenter.Such fit Include people, and other mammals with the example of the medicine agent or the experimenter of compositionss, including but not limited to little Mus, rat, Cavia porcelluss, rabbit, cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon and chimpanzee, particularly commercial important Animal or the animal that raises and train.In some embodiments, medicine agent or compositionss of the invention are can be formulated as suitable It is the experimenter of HLA-A2 for HLA antigens.
In another embodiment, the present invention also provides active component and is formulated as treating and/or prevent in preparation Cancer or tumor, including its recurrence after operation, medicine agent or pharmaceutical composition in purposes, the active component is selected from The following group：
The peptide of (a) present invention；
(b) in can expression-form coding peptide as disclosed herein polynucleotide；
C () presents the APC or allochthon of the peptide for having the present invention in its surface；With
The cytotoxic T cell of (d) present invention.
Or, the present invention further provides for treatment and/or prophylaxis of cancer or tumor active component, described activity into It is selected from the following group：
The peptide of (a) present invention；
(b) in can expression-form coding peptide as disclosed herein polynucleotide；
C () presents the APC or allochthon of the peptide for having the present invention in its surface；With
The cytotoxic T cell of (d) present invention.
Or, the present invention further provides a kind of pharmaceutical composition prepared for treatment and/or prophylaxis of cancer or tumor Or the method or technique of medicine agent, wherein the method or technique include with pharmacopedicss or physiologically acceptable carrier with The step of active component being selected from the group：
The peptide of (a) present invention；
(b) in can expression-form coding peptide as disclosed herein polynucleotide；
C () presents the APC or allochthon of the peptide for having the present invention in its surface；With
The cytotoxic T cell of (d) present invention.
In another embodiment, the present invention also provides a kind of preparation for treatment and/or prophylaxis of cancer or tumor Pharmaceutical composition or the method or technique of medicine agent, wherein the method or technique include mixed active composition with pharmacy or life The step of acceptable carrier of science, wherein described active component, are selected from the group：
The peptide of (a) present invention；
(b) in can expression-form coding peptide as disclosed herein polynucleotide；
C () presents the APC or allochthon of the peptide of the present invention in its surface；With
The cytotoxic T cell of (d) present invention.
In another embodiment, the present invention also provides the method for treatment and/or prophylaxis of cancer or tumor, wherein Methods described includes applying the step of being selected from following at least one active component to experimenter：
The peptide of (a) present invention；
(b) in can expression-form coding peptide as disclosed herein polynucleotide；
C () presents the APC or allochthon of the peptide of the present invention in its surface；With
The cytotoxic T cell of (d) present invention.
According to the present invention it has been found that comprising selected from SEQ ID NO:In 5,14,64,73,77,79,97,103 and 120 The peptide of aminoacid sequence is HLA-A2 restricted type epitope peptides, be therefore possible induction experimenter in for expression HLA-A2 and The candidate of the specific immunne response of the cancer of MPHOSPH1.In these peptides, more preferred example is with SEQ ID NO:The peptide of 120 aminoacid sequence, the cancer which has been verified as with induction targeted expression HLA-A2 antigens and MPHOSPH1 are thin The ability of the CTL of born of the same parents.Correspondingly, in preferred embodiments, medicine agent or pharmaceutical composition of the invention will include There is SEQ ID NO:The peptide of 120 aminoacid sequence or its modified forms, or the polynucleotide comprising the such peptide of coding.For Medicine agent or drug regimen comprising the polynucleotide of any one (polynucleotide i.e. of the invention) encoded in these peptides Thing is also such.
Cancer or tumor with the medicine agent or medicine composite for curing of the present invention is unrestricted, including wherein table Up to any cancer of (such as overexpression) MPHOSPH1, the example of cancer include but is not limited to bladder cancer, breast carcinoma, cervical cancer, Cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.
The medicine agent or pharmaceutical composition of the present invention can also be containing for example other have in addition to above-mentioned active component Induction is for the peptide of the ability of the CTL of cancerous cells, other polynucleotide of other peptides, other peptides described in presentation described in coding Other cells, etc..Such " other " there is induction to include, but are not limited to cancer for the peptide of the ability of the CTL of cancerous cells Specific antigen（The TAA for for example having identified）.
If necessary, medicine agent or pharmaceutical composition of the invention can optionally include other therapeutic substances as volume Outer active component, if the material do not suppress the present invention active component (peptide for example of the invention, polynucleotide, allochthon, APC, CTL) antitumous effect.For example, preparaton may include anti-inflammatory substance, analgesics, chemotherapeutics, such.Except medicine Outside other therapeutic substances in thing itself, the present invention medicine can with one or more other pharmacotoxicological effect agent or Compositionss are serially or simultaneously applied.The amount of pharmacotoxicological effect agent or compositionss depend on the pharmacotoxicological effect agent that for example used or The type of compositionss, the disease that is treated and the scheduling that applies and path.
It will be appreciated by those skilled in the art that in addition to the component for specifically referring to herein, the medicine effect of the present invention Agent or pharmaceutical composition may include that the conventional other materials in this area related to the preparaton type for being discussed (for example, are filled Agent, binding agent, diluent etc.).
In one embodiment of the invention, medicine agent or pharmaceutical composition of the invention can be packaged in product In, such as the disease to be treated containing can be used for treatment（Such as cancer）Pathological condition material test kit.Product May include the container and label of any medicine agent of the present invention or pharmaceutical composition.Suitable container include bottle, phial and Test tube.Container can be made with multiple material, such as glass or plastics.Label on container is noted that the agent or compositionss are used for Treatment or prophylactic one or more situation.Label also can indicate whether guidance with regard to administration etc..
In addition to above-described container, the test kit including medicine agent of the present invention or pharmaceutical composition can also be optional Second container is further included, pharmaceutically acceptable diluent is wherein housed.It can be further included from business and user's position Desired other materials are seen, including other buffer, diluent, filter, syringe needle, syringe and the packaging of operation instruction is loaded with Inset.
If desired, medicine agent or pharmaceutical composition can be packaged in medicated bag or dispenser device, the medicated bag Or dispenser device can be equipped with one or more unit dosage forms containing active component.For example, medicated bag may include metal or plastics Paper tinsel, such as blister pack.Medicated bag or dispenser device can have administration description.
(1) containing peptide as active component pharmaceutical composition：
The peptide of the present invention can be directly applied as medicine agent or pharmaceutical composition, or if necessary, by normal Advise compound method to prepare.In the later case, in addition to the peptide of the present invention, can also optionally include being generally used for medicine Carrier, excipient, etc., be not particularly limited.The example of examples of such carriers includes but is not limited to aquesterilisa, normal saline, phosphorus Phthalate buffer, culture fluid, etc..In addition, drug substance, medicine agent or pharmaceutical composition can when necessary containing stable Agent, suspension, preservative, surfactant etc..The medicine agent or pharmaceutical composition of the present invention can be used for anticancer purpose.
The peptide of the present invention can be prepared into combination, and which includes two or more peptides of the present invention, to induce CTL in vivo.Peptide Cocktail can be formed, or can be conjugated using standard technique each other.For example, it is possible to each chemistry of peptides connection or expression are become Single fused polypeptide sequence, the sequence can be with one or several aminoacid as joint (such as lysine joint： K.S.Kawamura et al.J.Immunol.2002,168:5709-5715).Each peptide in combination can be identical or Different.By applying the peptide of the present invention, peptide is illustrated on APC with high density by HLA antigens, is then induced and is shown The CTL that the complex specificity formed between peptide and HLA antigens reacts.Or, APC can be separated (for example from experimenter DC), then stimulate them with the peptide of the present invention, obtain the APC for showing any peptide of the present invention on its cell surface.Can CTL is induced in subject so that these APC are administered to experimenter again, as a result, can be improved for tumor mutually inside the Pass The aggressivity of skin.
Peptide including any present invention can also be wrapped as the medicine agent of the present invention or pharmaceutical composition of active component Adjuvant is included, effectively to set up cellular immunization, or, medicine agent of the present invention or pharmaceutical composition can be with other activity Composition is applied together, and they can be applied by being configured to granule.Adjuvant refer to the egg with immunologic competence White matter is together（Or order）Strengthen any compound, material or the compositionss of the immunne response for being directed to the protein during administration.Can Those (Clin Microbiol Rev 1994,7 described in document are included with the adjuvant that applies:277-89).Exemplary assistant Agent include but is not limited to Aluminium phosphate, aluminium hydroxide, Alumen, cholera toxin, salmonella toxin, incomplete Freund's adjuvant (IFA), Complete Freund's adjuvant (CFA), ISCOMatrix, GM-CSF, CpG, O/W emulsion etc., but not limited to this.
In addition, can be conveniently used liposome formulation agent, wherein peptide is bound to the particle formulation of the pearl of several micron diameters The preparaton of agent and wherein lipid binding to peptide.
In another embodiment of the present invention, peptide of the invention is also used as the form administration of officinal salt.Institute The preferred embodiment for stating salt includes the salt and the salt of amine of salt and organic base with alkali-metal salt and metal, with organic acid (second Acid, formic acid, propanoic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid etc. Deng) salt and salt with mineral acid (hydrochloric acid, phosphoric acid, hydrobromic acid, sulphuric acid etc.).As used in this article, phrase " officinal salt " Salt as referring to, they retain the biological effectiveness of compound and property, and are by anti-with inorganic or organic acid or alkali Answer and obtain.
In some embodiments, of the invention medicine agent or pharmaceutical composition include causing (prime) CTL into Point.Have been acknowledged that lipid is the material that can cause the CTL for virus antigen in vivo.For example, palmitic acid residues can be connected The ε-and alpha-amido of lysine residue is connected to, the peptide of the present invention is then attached to.Then the esterified peptide can be in micelle or granule In directly apply, mix liposome, or emulsifying in adjuvant.As another example that lipid causes CTL responses, escherichia coli (E.coli) lipoprotein, such as three palmityl-S- glyceryls cysteinyl seryls-serine (P3CSS), when with suitable When peptide is covalently attached, can be used to cause CTL (to see, for example, Deres et al., Nature 1989,342:561-4).
The example of suitable application process includes, but is not necessarily limited to, and oral, Intradermal, subcutaneous, intramuscular, bone are interior, abdomen Film and intravenous injection etc., and systemic application or local application to target site nearby (i.e. direct injection).Apply and can pass through Single administration is implementing, or is strengthened by repeatedly applying.The dosage of peptide of the present invention can be according to disease to be treated, patient Age, weight, the method etc. that applies appropriately adjusted, typically 0.001mg to 1,000mg, such as 0.01mg are extremely 100mg, such as 0.1mg to 10mg, and once can be applied once to every minority moon with the administration of every minority day.People in the art Member is readily selected suitable and optimum dosage.
(2) containing polynucleotide as active component pharmaceutical composition：
The present invention medicine agent or pharmaceutical composition also can contain in can expression-form coding disclosed herein Peptide polynucleotide.Herein, phrase " in can expression-form " means that polynucleotide can be when cell is imported The polypeptide of inducing antitumor immunity power is expressed as in vivo.In an exemplary embodiment, polynucleotide interested Nucleotide sequence includes regulating element necessary to polynucleotide expression.It is to realize stably inserting target cell that polynucleotide can have (description with regard to homologous recombination box carrier see, for example, Thomas KR＆Capecchi MR, Cell for configuration needed for genome 1987,51:503-12).Referring further to such as Wolff et al., Science 1990,247:1465-8;United States Patent (USP) No.5, 580,859;5,589,466;5,804,566;5,739,118;5,736,524;5,679,647;And WO98/04720).It is based on The example of the delivery technology of DNA includes " naked DNA ", Yi Hua（Bupivacaine, polymer, peptide-mediated）Delivery, cation lipid Complex and granule are mediated（" particle gun "）Or pressure-mediated delivery (see, for example, United States Patent (USP) No.5,922,687).
The peptide of the present invention can be also expressed with virus or bacteria carrier.The example of expression vector includes attenuated virus host, Such as cowpox or fowl pox.This method is directed to use with the nucleotide sequence that encoded peptide expressed by such as vaccinia viruss as carrier. After host is imported, recombined vaccinia virus expression expression immunogenic peptide, and thus cause immunne response.Can be used for immunity inoculation The vaccina vectors of scheme and method are recorded in such as United States Patent (USP) No.4,722,848.Another kind of carrier be bacillus calmette-guerin vaccine (BCG, Bacille Calmette Guerin).BCG carriers are recorded in Stover et al., Nature 1991,351:456-60.Have Many kinds can be used for therapeutic administration or other carriers of immunity inoculation are readily apparent that, such as adenoviruss and adeno associated viruss Carrier, retroviral vector, salmonella typhi (Salmonella typhi) carrier, detoxification anthrax toxin vectors, such as Such.See, for example, Shata et al., Mol Med Today 2000,6:66-71;Shedlock et al.,J Leukoc Biol 2000,68:793-806;Hipp et al.,In Vivo 2000,14:571-85.
It can be direct that polynucleotide are delivered into patient, wherein make experimenter be directly exposed to carry polynucleotide Carrier, or indirectly, wherein then cell transplantation is entered to suffer from vitro with polynucleotide transformed cells interested first Person.Both methods are referred to as internal and ex vivo gene therapy.
With regard to gene therapy method general summary referring to Goldspiel et al., Clinical Pharmacy 1993,12:488-505;Wu and Wu,Biotherapy1991,3:87-95;Tolstoshev,Ann Rev Pharmacol Toxicol 1993,33:573-96;Mulligan,Science 1993,260:926-32;Morgan&Anderson,Ann Rev Biochem 1993,62:191-217;Trends in Biotechnology 1993,11(5):155-215.? eds.Ausubel et al.,Current Protocols in Molecular Biology,John Wiley&Sons,NY, 1993;And Krieger, in Gene Transfer and Expression, A Laboratory Manual, Stockton The known method in the recombinant DNA technology field described in Press, NY, 1990 can be applicable to the present invention.
As administration for peptides, the administration of polynucleotide can pass through in oral, Intradermal, subcutaneous, intravenouss, intramuscular, bone, And/or peritoneal injection etc. is carrying out, and by systemic application or local application to target site.Apply and can pass through list Secondary administration is implementing, or is strengthened by repeatedly applying.The polynucleotide conversion of suitable carrier or encoded peptide of the present invention Cell in the dosage of polynucleotide can be according to disease to be treated, the age of patient, weight, the method etc. applied just When being adjusted, typically 0.001mg to 1000mg, such as 0.01mg to 100mg, such as 0.1mg to 10mg, and can be with every A couple of days applies and once applies once to per several months.Those skilled in the art can appropriately select suitable dosage.
X. using the method for peptide, allochthon, APC and CTL：
The peptide and polynucleotide of the present invention can be used to preparing or inducing APC and CTL.The allochthon and APC of the present invention also may be used For inducing CTL.And for induction for cancer or the immunne response of tumor.The peptide, polynucleotide, allochthon and APC Can be applied in combination with any other compound, as long as other compound does not suppress their CTL inducibilities.Therefore, The medicine agent or pharmaceutical composition of any previously described present invention is used equally to induce CTL.In addition, those bags Include peptide and polynucleotide can also be used for induce APC, as explained below.
(1) method of the inducing antigen in delivery cell (APC)：
The invention provides the method for inducing the APC with CTL inducibilities using peptide of the invention or polynucleotide.
The method of the present invention includes the step of making APC contact in vitro, in vitro or in vivo with the peptide of the present invention.For example, wrap The method for including the step of contacting APC with peptide in vitro may include following step：
a：APC is collected from experimenter；With
b：The APC of step a is made to contact with the peptide of the present invention.
APC is not limited to particular kind of cell, and including DC, Langerhans cell, macrophage, B cell and activation T cell, it is known that the antigen of their presenter protein properties on their cell surface, so as to be recognized by lymphocyte.Excellent Selection of land, can use DC, because DC is that there is in APC most strong CTL inducibilities.Any peptide of the present invention individually can make With or the peptide of the invention with other or derived from MPHOSPH1 outside the inducible peptides of CTL of TAA be applied in combination.
On the other hand, when the peptide of the present invention is administered to experimenter, APC touches the peptide in vivo, as a result, The APC with CTL inducibilities is induced in the body of experimenter.Therefore, the method for the present invention includes applying the peptide of the present invention For experimenter inducing the APC with CTL inducibilities in the subject.Similarly, when will be in shape can be expressed When the polynucleotide of the present invention of formula are applied to experimenter, the peptide of the present invention is expressed in vivo and is contacted with APC, as a result, received The APC with CTL inducibilities is induced in the body of examination person.Therefore, the method for the present invention is may also comprise many of the present invention Nucleotide is applied to experimenter to induce the APC with CTL inducibilities in the subject.Term " effable shape Formula " described above " IX. pharmaceutical compositions, (2) containing polynucleotide as active component pharmaceutical composition " part.
Additionally, the method for the present invention may include the polynucleotide of the present invention are imported APC to induce with CTL inducibilities APC.For example, the method may include following step：
a：APC is collected from experimenter；And
b：The polynucleotide for importing coding peptide of the present invention import APC.
Step b can be implemented as described in " VI. antigen-presenting cells " part above.
Or, the invention provides a kind of for preparing specificity induction for the antigen of the CTL activity of MPHOSPH1 be in The method of delivery cell (APC), wherein methods described may include one of following step：
A () contacts APC and the peptide of the present invention in vitro, in vitro or in vivo；With
B () imports the polynucleotide for encoding the peptide of the present invention in APC.
Or, the present invention is provided to the method that induction has the APC of CTL inducibilities, wherein methods described includes selecting From following step：
A () contacts APC and the peptide of the present invention；
B the polynucleotide for encoding the peptide of the present invention are imported APC by ().
The method of the present invention can be implemented in vitro, in vitro in vivo.Preferably, the method for the present invention can in vitro or In vitro enforcement.The APC for induction with the APC of CTL inducibilities can advantageously be the APC for expressing HLA-A2 antigens.This The APC of sample can be public with this area by the peripheral blood lymphocytes (PBMC) obtained from the experimenter that HLA antigens are HLA-A2 Prepared by the method that knows.The APC induced with the method for the present invention can be the peptide and HLA antigens for presenting the present invention in its surface The APC of the complex of (HLA-A2 antigens).When the APC induced by the method for the present invention to be administered to experimenter to receive at this When in examination person, induction is directed to the immunne response of cancer, the experimenter is preferably the same experimenter originated by APC.However, tested Person can be differently configured from APC donors, as long as experimenter is identical with the HLA types of APC donors.
In another embodiment, the present invention is provided to induction have CTL inducibilities APC used in effect Agent or compositionss, and such agent or compositionss include the peptide or polynucleotide of one or more present invention.
In another embodiment, the present invention provides the polynucleotide of the peptide or coding for said peptides of the present invention in preparation preparation It is for inducing the purposes in the compositionss of APC.
Or, the present invention also provides the polynucleotide of the peptide or coding for said peptides of the present invention, and which is used for, in induction, there is CTL Used in the APC of inducibility.
(2) method for inducing CTL：
Present invention also offers the method for inducing CTL using the peptide of the present invention, polynucleotide or allochthon or APC.
Present invention also offers using polynucleotide for encoding two TCR subunits or encoding each TCR subunit Multiple polynucleotide come the method that induces CTL, wherein described TCR is capable of identify that (in conjunction with) presents this on cell surface Bright peptide and the complex of HLA antigens.Preferably, the method for the induction CTL may include at least one step being selected from the group：
A) CD8 positive T cells are made to contact with antigen-presenting cell and/or allochthon, the antigen-presenting cell and external Body presents the complex formed between HLA antigens and the peptide of the present invention in its surface；With
B) polynucleotide of two TCR subunits of coding are imported in CD8 positive T cells or encode each TCR Asia Multiple polynucleotide of unit, wherein described TCR are capable of identify that (in conjunction with) presents the peptide and HLA of the present invention on cell surface The complex of antigen.
After peptide, polynucleotide, APC or allochthon as the present invention is administered to experimenter, induce in the body of experimenter Go out CTL, and the immunne response of the cancerous cell of targeting coding MPHOSPH1 strengthens.Therefore, the method for the present invention is may include this The step of peptide of invention, polynucleotide, APC or allochthon are applied to experimenter.
Or, can also pass through in vitro or use them to induce CTL in vitro, and after induction CTL, can be by activation CTL returns experimenter.For example, the method may include following step：
a：APC is collected from experimenter；
b：APC with peptide contact procedure a；And
c：The APC of step b is co-cultured with CD8 positive cells.
The APC that will be co-cultured with CD8 positive cells in above step c can also pass through the polynucleotide comprising the present invention Gene transfer enters APC above as described in " VI. antigen-presenting cells " part to prepare, but the invention is not restricted to this, and Therefore cover the APC of the complex that any effective on the surface presentation HLA antigens and peptide of the present invention are formed.
The allochthon of the complex for being optionally usable on its surface presenting HLA antigens and peptide of the present invention formation is substituting Above-mentioned APC.That is, the present invention may include the external of the complex for forming presentation HLA antigens in its surface and peptide of the present invention The step of body is co-cultured.Such allochthon can be prepared by the method described in " V. allochthons " part above.
Can preferably be to present peptide of the invention and HLA-A2 in its surface for inducing the APC or allochthon of CTL The APC or allochthon of the complex of antigen.
In addition, the polynucleotide or coding for importing two TCR subunits of coding in CD8 positive T cells can be passed through Inducing CTL, wherein described TCR is capable of identify that (in conjunction with) is presented in cell surface to multiple polynucleotide of each TCR subunit On the present invention peptide and HLA antigens complex.Such transduction can be such as institute in " VIII.T cell receptors (TCR) " part above State to implement.
The method of the present invention can be implemented in vitro, in vitro in vivo.Preferably, the method for the present invention can in vitro or In vitro enforcement.For inducing the CD8- positive T cells of CTL can be by obtaining PBMC methods well known in the art from experimenter Prepare.In preferred embodiments, the donor of CD8- positive T cells can be the experimenter that HLA antigens are HLA-A2.With this The CTL that the method for invention is induced can present the CTL for having the peptide with the complex of HLA antigens of the present invention in its surface. Should for the immunity of cancer with induction in the experimenter when the CTL induced by the method for the present invention is administered to experimenter When answering, the experimenter is preferably the same experimenter originated by CD8- positive T cells.However, experimenter can be differently configured from CD8- Positive T cell donor, as long as experimenter is identical with the HLA types of CD8- positive T cell donors.
In addition, the invention provides a kind of manufacture for induce CTL medicine agent or pharmaceutical composition method or The step of technique, wherein the method or technique include the peptide and pharmaceutically acceptable carrier for mixing or preparing the present invention.
In another embodiment, the present invention is provided to the agent of induction CTL or compositionss, wherein described agent Or peptide of the compositionss comprising one or more present invention, the polynucleotide of one or more present invention or one or more send out Bright APC or allochthon.
In another embodiment, the present invention provides peptide of the invention, polynucleotide or APC or allochthon and matches somebody with somebody for preparing Be made as induce CTL agent or compositionss in purposes.
Or, the present invention provides peptide, polynucleotide or the APC or allochthon of the present invention to be used for inducing CTL.
(3) method for inducing immunne response：
Additionally, the invention provides the method for inducing the immunne response for the disease for being related to MPHOSPH1.The disease of consideration Disease includes that cancer, its example include but is not limited to bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, stomach Cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.
The method of the present invention may include to apply containing any peptide of the present invention experimenter or encode their polynucleotide The step of agent or compositionss.The method of the present invention can also cover apply present have any the present invention peptide allochthon or APC.With regard to details referring to " IX. pharmaceutical compositions " item, the medicine agent or pharmaceutical composition for particularly describing the present invention is made Part for the purposes of vaccine.In addition, the allochthon and APC for the method for present invention induction immunne response is described in detail in (1) of literary " V. allochthons ", " VI. antigen-presenting cells (APC) " and " methods of the X. using peptide, allochthon, APC and CTL " and (2) part.
In preferred embodiments, it is HLA-A2 that the experimenter for being treated with the method for the present invention can be HLA antigens Experimenter.
Present invention also offers manufacturing for inducing the method or work of the medicine agent or pharmaceutical composition of immunne response The step of skill, wherein the method may include peptide or polynucleotide and the pharmaceutically acceptable carrier for mixing or preparing the present invention.
Or, the method for the present invention may include to apply the vaccine of the present invention or the step of pharmaceutical composition, the vaccine or Pharmaceutical composition includes：
The peptide of (a) present invention；
The nucleic acid (polynucleotide) for encoding such peptide as disclosed herein of (b) in effable form；
C () presents the APC or allochthon of the peptide for having the present invention in its surface；Or
The cytotoxic T cell of (d) present invention.
In the linguistic context of the present invention, the cancer for expressing MPHOSPH1 can be treated with these active component.The cancer Example includes but is not limited to bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymph Tumor, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.Thus, in vaccine or medicine group that administration includes the active component Before compound, the normal cell phase of the MPHOSPH1 expressions in cell or tissue to be treated and same organs is preferably confirmed Than whether enhancing.Therefore, in one embodiment, the invention provides expressing the side of the cancer of MPHOSPH1 for treatment Method, the method may include following step：
I) the MPHOSPH1 expressions in the cell or tissue obtained from the experimenter with cancer to be treated are determined；
Ii) the MPHOSPH1 expressions are compared with normal control；And
Iii) experimenter to having the cancer of overexpression MPHOSPH1 compared with normal control applies at least one and is selected from The composition of steps described above (a) to (d).
Or, the present invention also provides the vaccine or medicine group for including at least one composition selected from (a) mentioned above to (d) Compound, its are used for the experimenter to the cancer with overexpression MPHOSPH1 and apply.In other words, invention further provides using In the method for experimenter of the identification with MPHOSPH1 polypeptides of the present invention to treat, methods described includes determining from experimenter's Cancerous cell or tissue in MPHOSPH1 expressions the step of, wherein the level is compared with the normal control values of the gene Raise and indicate that the experimenter there may be the cancer that can be treated with MPHOSPH1 polypeptides of the present invention.The identification of the present invention will be treated The method of the experimenter of cancer is described in greater detail below.
Any cell or tissue from experimenter is used equally to determine MPHOSPH1 expressions, as long as which includes target MPHOSPH1 transcription or translation product.The example of appropriate samples includes but are not limited to bodily tissue and body fluid, such as blood, Expectorant and urine.Preferably, the cell or tissue sample from experimenter contains such cell colony, and the colony includes that epithelium is thin Born of the same parents, more preferably cancerous epithelial cell or from suspect for carcinous tissue epithelial cell.Further, if necessary, can be from institute The bodily tissue for obtaining and cell described in purification in body fluid, are then used as the sample from experimenter.
The experimenter that need to be treated with this method is preferably mammal.Exemplary mammal includes but are not limited to example Such as the mankind, non-human primates, mice, rat, dog, cat, horse and cattle.
According to the present invention it is possible to determine the expression of MPHOSPH1 in the cell or tissue obtained from experimenter.Table Can be in transcription up to level（Nucleic acid）Product level determines, using means known in the art.For example, the mRNA of MPHOSPH1 can By hybridizing method（For example, Northern hybridization）Using probe quantitative.Detection can be implemented on chip, array etc..To inspection For surveying the expression of MPHOSPH1, the use of array is probably preferred.Those skilled in the art can utilize MPHOSPH1's Sequence information prepares above-mentioned probe.For example, the cDNA of MPHOSPH1 can be used as probe.If desired, the probe can be used closing Suitable label comes labelling, such as dyestuff, fluorescent material and isotope, and the expression of the gene can be used as being hybridized Label intensity detection.
Additionally, transcription product (for example, the SEQ ID NO of MPHOSPH1:125) detection method based on amplification can also be passed through （For example, RT-PCR）Using primers.Such primer can be prepared based on the obtained sequence information of the gene.
Specifically, the probe or primer used by this method is under stringent condition, medium stringency condition or low stringency condition Hybridize with the mRNA of MPHOSPH1.As used herein, phrase is " strict（Hybridization）Condition " refers to such condition, at this Under the conditions of, probe or primer will with its target sequence hybridization, but not with other sequence hybridizations.Stringent condition is to rely on sequence, And in different environments can be different.The specific hybridization of longer sequence is observed compared with shorter sequence at relatively high temperatures. Usually, the temperature of stringent condition selects bit sequencing to be listed in the heat fusion joint under the ionic strength and pH of restriction (Tm) low by about 5 Degree Celsius.Tm be (under the ionic strength, pH and nucleic acid concentration for limiting) have under poised state 50% with its target complement sequence The temperature that probe is hybridized with target sequence.Because target sequence is typically present in excess, therefore in Tm, balance, 50% probe is occupied. Typically, stringent condition can be such：Wherein salinity is less than about 1.0M sodium ion, typically about 0.01-1.0M sodium Ion (or other salt), pH7.0-8.3, temperature are at least big for shorter probe or primer (such as 10-50 nucleotide) About 30 degrees Celsius, be at least about 60 degrees Celsius for longer probe or primer.Stringent condition can also be gone surely by adding Earnest matter, such as Methanamide, realize.
The probe or primer of the present invention are typically the oligonucleotide of substantially purification.Oligonucleotide is typically comprised so Nucleotide sequence region, its under strict conditions with include MPHOSPH1 sequences nucleic acid at least about 2000,1000, 500,400,350,300,250,200,150,100,50, or 25 continuous sense strand nucleotide sequences, or including The antisense strand nucleotide sequence of the nucleic acid of MPHOSPH1 sequences, or the naturally occurring mutant hybridization of these sequences.Especially Be, for example, in preferred embodiments, it is possible to use length for 5-50 oligonucleotide expand as primer to be detected Gene.It is highly preferred that particular size, the usually oligonucleotide probe or primer of length 15-30b can be used detecting The mRNA or cDNA of MPHOSPH1 genes.Size can be at least 10 nucleotide, at least 12 nucleotide, at least 15 nucleoside The size of acid, at least 20 nucleotide, at least 25 nucleotide, at least scope of 30 nucleotide, and probe and primer can be with For 5-10 nucleotide, 10-15 nucleotide, 15-20 nucleotide, 20-25 nucleotide and 25-30 nucleotide.Excellent In the embodiment of choosing, the length of oligonucleotide probe or primer can be selected from 15-25.Using such oligonucleotide probe Or the mensuration program of primer detection gene, device or reagent are known (such as oligonucleotide microarrays or PCR).In these surveys In fixed, probe or primer can also include label or joint sequence.In addition it is possible to use detectable label or to be captured Affinity ligand is modifying probe or primer.Or, in the detection program based on hybridization, it is also possible to using length for hundreds of (e.g., from about 100-200) base is used for probe (for example to the polynucleotide of thousands of (e.g., from about 1000-2000) bases Northern traces are determined or cDNA microarray analysis).
Or, translation product can be detected to carry out the diagnosis of the present invention.For example, it may be determined that MPHOSPH1 albumen (SEQ ID NO:126) or its immunologic competence fragment amount.Measure includes immunoassay side as the method for the protein content of translation product Method, antibody of such method using albumen described in specific recognition.Antibody can be monoclonal or polyclone.And, antibody Any fragment or modification (such as chimeric antibody, scFv, Fab, F (ab ')2, Fv etc.) be used equally to detect, as long as the fragment or warp Modified antibodies retain the binding ability to MPHOSPH1 albumen.The present invention also provide such peptide for the present invention and Antibody and its fragment.The method for detecting the antibody of albumen for preparing these types be well-known in the art, and These antibody and their equivalent can be prepared using any method in the present invention.
The method of the expression of the translation product detection MPHOSPH1 genes as another kind based on MPHOSPH1, can profit Its intensity for dyeing is measured with the antibody for MPHOSPH1 albumen by immunohistochemical analysis.This means, in this measurement In, strong dyeing shows that the protein presence/level increases, and shows the high expression level of MPHOSPH1 genes simultaneously.
Target gene (such as MPHOSPH1 genes) in for cancerous cell, if which is than the control level (example of target gene Such as the level in normal cell) increased if such as 10%, 25% or 50%, or 1.1 times are increased to above, more than 1.5 times, surpass 2.0 times are crossed, more than 5.0 times, more than 10 times or more, then it is considered that its expression in cancerous cell increased.
Control level can be determined with cancerous cell simultaneously, be received from morbid state (suffer from cancer or be not suffering from cancer) is known using previous The sample that examination person (several) collects and preserves.In addition, obtaining from the non-cancerous area of the organ with cancer to be treated Normal cell can be used as normal control.Or, control level can be by statistical method, according to previously determined by analysis The result obtained from the MPHOSPH1 gene expression doses of the sample of experimenter known to morbid state is determined.Enter one Step, control level can be from the expression pattern data base of the cell that had previously tested.And, according to a side of the present invention Face, can be by the expression of MPHOSPH1 genes in biological sample and the multiple control level ratios determined from multiple reference samples Compared with.It is preferably used from the reference sample determination from the organization type similar to the organization type of the sample for being derived from experimenter Control level.It is further preferred that the standard using MPHOSPH1 gene expression doses in the colony with known morbid state Value.Standard value can be obtained by any method known in the art.For example, the +/- 2S.D. of the meansigma methodss or +/- 3S.D. of meansigma methodss Scope can serve as standard value.
In the linguistic context of the present invention, the control level determined from the biological sample of known non-cancerous is referred to as " normal control water Flat ".On the other hand, if control level is determined from carcinous biological sample, " carcinous control level " can be referred to as.Sample table reaches water Flat difference between control level can relative known expression will not be with the control of the cancer of cell or non-cancer state change The standardization in addition of the expression of nucleic acid (such as house-keeping gene).The crt gene of example include but not limited to, and β-flesh moves egg In vain, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein P1.
Normal expression level is compared when the expression of MPHOSPH1 genes to increase, or with carcinous control level phase Like/equivalent, then it is with cancer to be treated that experimenter is diagnosable.
Present invention also offers whether a kind of (i) diagnosis experimenter is doubtful with cancer to be treated, and/or (ii) is selected The method that experimenter carries out treatment of cancer, the method may include following step：
A) MPHOSPH1 is determined from the cancerous cell or tissue for suspecting experimenter's acquisition with cancer to be treated Expression；
B) expression of MPHOSPH1 is compared with normal control values；
If if c) expression of MPHOSPH1 is raised compared with the normal control level, be diagnosed as having by experimenter and wanting The cancer for the treatment of；And
If if d) experimenter is diagnosed as there is cancer to be treated in step c), selecting experimenter to carry out cancer and controlling Treat.
Or, such method may include following step：
A) determine in the table from MPHOSPH1 in the cell or tissue for suspecting experimenter's acquisition with cancer to be treated The level of reaching；
B) expression of MPHOSPH1 is compared with carcinous control level；
If if c) expression of MPHOSPH1 is similar or equivalent with carcinous control level, experimenter is diagnosed as have Cancer to be treated；And
If if d) experimenter is diagnosed as there is cancer to be treated in step c), selecting experimenter to carry out cancer and controlling Treat.
Present invention also offers for diagnose or determine with doubtful suffer from or risky generation can use the present invention The diagnostic kit of the experimenter of cancer of the MPHOSPH1 polypeptides to treat, which also can be used for evaluating and/or monitors cancer immunity Effect of therapy or the suitability.Preferably, the cancer includes but is not limited to bladder cancer, breast carcinoma, cervical cancer, bile duct cell Cancer, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms.More particularly, The test kit can preferably include at least one reagent for detecting MPHOSPH1 gene expressions in the cell for being derived from experimenter, institute State reagent to be selected from the group：
A () is used for the reagent of the mRNA for detecting MPHOSPH1 genes；
B () is used for the reagent for detecting MPHOSPH1 albumen or its immunologic competence fragment；With
C () is used for the reagent of the biologic activity for detecting MPHOSPH1 albumen.
The reagent for being adapted to detect for MPHOSPH1 gene mRNAs may include the core for specifically binding or recognizing MPHOSPH1 mRNA Acid, such as has the oligonucleotide with a part of complementary sequence of MPHOSPH1 mRNA.It is right that the example of this class oligonucleotide has The specific primers of MPHOSPH1 mRNA and probe.This class oligonucleotide can be prepared based on method well-known in the art.Such as Fruit needs, for detecting that the reagent of MPHOSPH1 mRNA can be immobilized in solid matrix.In addition, can in the test kit Reagent including more than one detection MPHOSPH1 mRNA.
On the other hand, the reagent for being adapted to detect for MPHOSPH1 albumen or its immunologic competence fragment may include to be directed to MPHOSPH1 albumen or the antibody of its immunologic competence fragment.The antibody can be monoclonal or polyclone.Further, appoint The fragment of what antibody or modification（For example, chimeric antibody, scFv, Fab, F (ab ')2, Fv etc.）Can be employed as the examination Agent, as long as the fragment or modified antibody retain the binding ability with MPHOSPH1 albumen or its immunologic competence fragment.For Detection albumen prepares the method for this antibody-like and is well known in the art, and can be prepared using any method in the present invention Above-mentioned antibody and its equivalent.Further, the antibody can pass through to be directly connected to or indirect labelling with the molecule that can produce signal Technology is marked.The method of the combination of label and traget antibody and detection antibody and its target is well-known in this area , and the present invention can use any label and method.In addition, may include in the test kit more than one for detecting The reagent of MPHOSPH1 albumen.
The test kit can include more than one aforesaid reagent.The test kit can also include for combine be directed to The probe of MPHOSPH1 genes or for MPHOSPH1 peptides antibody solid matrix and reagent, for cultured cells culture Base and container, positive and negative control reagent and the second antibody for detecting the antibody for MPHOSPH1 peptides.Citing and The tissue sample that speech, never cancer or the experimenter with cancer are obtained can be used as useful contrast agents.The examination of the present invention Agent box can further include other from desired material for business or user perspective, including buffer, diluent, filter, pin Head, syringe and carry operation instructions（For example, written, tape, CD-ROM etc.）Packing list.These reagents etc Label can be put on comprising in a reservoir.Suitable container may include bottle, pipe-type bottles (vials) and test tube.The container is available more Plant material manufacture, such as glass or plastics.
In one embodiment of the invention, when the reagent is the probe for MPHOSPH1 mRNA, the examination Agent can be immobilized onto on solid matrix such as porous bar, to form at least one test position.The measurement or inspection of the porous bar Survey region and can include multiple positions, each include nucleic acid（Probe）.Test strip can also include the position of negative and/or positive control Put.Or, control site is can be located on the bars different from test strip.Optionally, different test positions can include different amounts of Immobilized nucleic acids, i.e. measure in first test position measure on larger and position below less.Add test sample it Afterwards, show that the number of detectable signal position provides the quantitative indication of the MPHOSPH1 mRNA amounts for existing in the sample.Described Test position is can be set to any properly detectable shape, typically across strip or the point-like of test strip width.
Test kit of the present invention can further include positive control sample or MPHOSPH1 standard sample.The institute of the present invention State positive control sample to pass through to collect MPHOSPH1 positives, subsequently determine its MPHOSPH1 level to prepare.Additionally, can The MPHOSPH1 albumen or polynucleotide of purification are added in the cell for do not express MPHOSPH1 to form the positive Or MPHOSPH1 standard sample.In the linguistic context of the present invention, the MPHOSPH1 of purification can be recombiant protein.For example, positive right The MPHOSPH1 levels of product are more than cutoff value in the same old way.
In one embodiment, invention further provides a kind of diagnostic kit, which is included by antibody of the present invention Albumen of specific recognition or part thereof albumen or its immunogenic fragments.
The example of the partial peptide of the protein of the present invention include at least 8 in the aminoacid sequence by albumen of the present invention, It is preferred that the polypeptide that 15, more preferably 20 continuous amino acids are constituted.Cancer can be by using the albumen of the present invention or peptide（Polypeptide） Detection sample（Such as blood, tissue）In antibody diagnosing.Described above is for preparing the peptide or protein of the present invention Method.
The method for diagnosing cancer of the present invention can pass through anti-MPHOSPH1 amount of antibody determined as outlined above and mutually tackle Difference between amount in product is carrying out in the same old way.If the cell or tissue of experimenter contains the expression product for the gene (MPHOSPH1) the antibody and amount of the anti-MPHOSPH1 antibody for measuring is more than cutoff value（With the level phase in normal control Than）If, then the experimenter is suspected with cancer.
In another embodiment, diagnostic kit of the invention may include the peptide and the HLA combined with it of the present invention Molecule.(such as Altman JD is established already using the appropriate method of antigenic peptide and HLA Molecular Detection Peptide-specific CTLs et al.,Science.1996,274(5284):94-6).Therefore, the complex that peptide of the present invention and HLA molecules are formed can be used for Detection method detecting specific for tumour antigen CTL, thus, it is possible to the recurrence and/or transfer of relatively early detection cancer.Further, it Can be used to select to be suitable for the therapeutic effect for including peptide of the present invention as the experimenter of the medicine of active component or assessing the medicine.
Especially, (Altman JD et al., Science.1996,274 (5284) see, for example, according to known method: 94-6), the oligomer complex of radiolabeled HLA molecules and peptide of the present invention, the such as tetramer can be prepared.For example can pass through Using the complex come the antigen-peptide specific CTL to being derived from the peripheral blood lymphocyte for suspecting the experimenter with cancer Quantitatively being diagnosed.
Invention further provides assessing the side of the immunological response of experimenter using peptide epitopes as described herein Method or diagnostic reagent.In one embodiment of the invention, HLA-A2 as herein described can be used to limit peptide come as reagent Assessment or the immunne response of prediction experimenter.Immunne response to be assessed is exempted from by making immunogen contact in vitro or in vivo Epidemic disease competent cell is inducing.In some embodiments, any may result in recognizes and the Peptide-specific CTL with reference to peptide epitopes Generation material or compositionss can be employed as the reagent.Peptide reagent is without the need for being used as the immunogen.For this alanysis Measurement system includes that relatively new technology develops, the such as dyeing of the tetramer, intracellular lymphokine and interferon release algoscopy, Or ELISPOT algoscopys.In preferred embodiments, periphery can be selected from for assessing the immunologically competent cell of immunne response Blood, peripheral blood lymphocyte (PBL) and PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC).Thin for collecting or separating such immunocompetence The method of born of the same parents is well known in the art.In the preferred embodiment for substituting, the immunologically competent cell bag that will contact with peptide reagent Include antigen-presenting cell, such as dendritic cell.
For example, peptide of the invention can be used for tetramer staining algoscopy, assessment PERIPHERAL BLOOD MONONUCLEAR CELL be exposed to swollen The presence of Peptide-specific CTL after oncocyte antigen or immunogen.HLA tetramer complex can be used to directly manifest antigen-specific Property CTL (see, for example, Ogg et al., Science 279:2103-2106,1998;With Altman et al, Science 174:94-96,1996) the frequency with measure Peptide-specific CTL colony in PERIPHERAL BLOOD MONONUCLEAR CELL sample.Using this The tetramer reagent of bright peptide can be as described below generating.
Make the peptide combined to HLA molecules in the presence of corresponding HLA heavy chains and B2M refolding generating three molecules Complex.In the complex, previously site of the through engineering approaches in protein is biotinylated the carboxyl terminal of heavy chain.So Afterwards, streptavidin is added to complex forming the tetramer being made up of three molecular complexes and streptavidin.By fluorescence The streptavidin of labelling, dyes to antigen-specific cellular using the tetramer.Then for example can be reflected by flow cytometry Determine cell.This alanysis can be used for diagnosis or prognosis purpose.The cell that is identified by the code can also be used for therapeutic purposes.
Present invention also offers the reagent for immune anamnestic response including peptide of the present invention (see, for example, Bertoni et al,J.Clin.Invest.100:503-513,1997 and Penna et al., J Exp.Med.174:1565-1570, 1991).For example, antigen-specific is analyzed to the Patient PBMC samples for being derived from the individuality with cancer to be treated using particular peptide The presence of property CTL.Blood sample containing mononuclearcell can be assessed as follows：Culture PBMC, and should with the peptide stimulation of the present invention Cell.Suitably after the culture period, the cell colony for expanding can be analyzed, for example, analyze CTL activity.
Peptide is alternatively arranged as effect that reagent is used for assessing vaccine.Can use example method is connect from vaccine to analyze as described above Plant the PBMC that immunogenic patient obtains.The HLA types of patient are determined, and selects to recognize allele specific present in the patient The peptide epitopes reagent of property molecule is analyzed.The immunogenicity of vaccine can pass through the presence of epitope specificity CTL in PBMC samples To indicate.The peptide of the present invention can be additionally used in preparing antibody, (see, for example, CURRENT using techniques well known PROTOCOLS IN IMMUNOLOGY,Wiley/Greene,NY;With Antibodies A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which can be used for examining as reagent Disconnected, detection or monitoring cancer.This antibody-like may include that the antibody for recognizing the peptide in HLA molecular backgrounds, i.e. binding peptide-MHC are combined The antibody of thing.
The peptide of the present invention and compositionss also have multiple other applications, there is described herein some of them.For example, the present invention Provide a kind of method for diagnosing or detect the disease being characterized with the amount of MPHOSPH1 immunogenic polypeptides.These methods The complex for being related to measure MPHOSPH1HLA binding peptides or MPHOSPH1 peptides with the formation of I classes HLA molecule is in biological sample Expression.The expression of the complex that peptide or peptide and I classes HLA molecule are formed can pass through to be surveyed with the combination spouse for peptide or complex Surely determining or detect.In a preferred embodiment, the combination spouse for peptide or complex can be identification and spy The antibody of the anisogamy peptide.Expression of the MPHOSPH1 in biological sample such as tumor biopsy also can be drawn using MPHOSPH1 Thing is tested by standard PCR amplification scheme.One example of tumor expression presented herein, and for MPHOSPH1 expansions The exemplary condition for increasing and the further disclosure of primer are found in WO2003/27322, are incorporated in which herein by quoting Hold.
Preferably, diagnostic method is related to make to contact to MPHOSPH1 peptide specifics from the detached biological sample of experimenter Agent is detecting presence of the MPHOSPH1 peptides in the biological sample.As used in this article, " contacting " means suitable Suitable condition（Such as concentration, temperature, time, ionic strength）Lower placement biological sample makes its close enough agent, with Specificity present in permissive effect agent and biological sample between MPHOSPH1 peptides interacts.Usually, agent connects The condition of tactile biological sample be skilled addressee will appreciate that promoted biological sample in molecule be associated with thing （Such as protein is complementary to serial correlation thing with its receptor related compounds, antibody and its proteantigen related compounds, nucleic acid）It Between specificity interact condition.For promoting molecule to be associated with the optimal conditionss of the interaction of the specificity between thing It is recorded in United States Patent (USP) No.5 for licensing to Low et al., 108,921, its content is incorporated to herein by quoting.
The diagnostic method of the present invention can be implemented in vivo and/or in vitro.Thus, biological sample in the present invention may be used It is located at inner or in vitro.For example, biological sample can be in-vivo tissue, and can use to MPHOSPH1 immunogenic polypeptides Specific agent is detecting the presence in the tissue of this quasi-molecule.Or, can collect in vitro or separate biological sample （Such as blood sample, tumor biopsy, tissue extract）.In an especially preferred embodiment, biological sample can be with It is the sample containing cell, the sample containing tumor cell that more preferably collects from the experimenter that will be diagnosed or treat.
Or, diagnosis can be used allows by the dyeing of fluorescein-labeled HLA multimeric complexes, direct quantitative is anti- The method of former specific T-cells carrying out (such as Altman, J.D.et al., 1996, Science 274:94;Altman, J.D.et al.,1993,Proc.Natl.Acad.Sci.USA90:10330).Additionally provide the dyeing to intracellular lymphokine Algoscopy or ELISPOT algoscopys is discharged with interferon-γ.Polymer dyeing, the dyeing of intracellular lymphokine and ELISPOT are determined Method all show sensitiveer at least 10 times than more conventional algoscopy (Murali-Krishna, K.et al., 1998, Immunity 8:177;Lalvani,A.et al.,1997,J.Exp.Med.186:859;Dunbar,P.R.et al.,1998, Curr.Biol.8:413).Pentamer (such as US2004-209295A), Dextramers (such as WO02/ can also be used And Streptamers (such as Nature medicine 6.631-637 (2002)) 072631).
Correspondingly, in some embodiments, the present invention provides one kind and has been applied at least one for diagnosing or evaluating The method of the immunne response of the experimenter of the MPHOSPH1 peptides of the present invention, methods described comprise the steps：
A () makes immunogen connect with tool immunologically competent cell under conditions of being suitable for inducing the CTL to immunogens Touch；
The induced levels of the CTL of induction in (b) detection or determination step (a)；With
C the immunne response of the experimenter is associated by () with the CTL induced levels.
In the linguistic context of the present invention, immunogen preferably includes at least one of following：A () is selected from SEQ ID NO:5,14, The MPHOSPH1 peptides of 64,73,77,79,97,103 and 120 aminoacid sequence, and (b) have the aminoacid sequence, wherein The peptide that the aminoacid has been modified through 1,2 or more aminoacid replacement.Meanwhile, it is suitable for induction immunogens The condition of CTL is well known in the art.For example, it is possible in the presence of immunogen, culture has immunologically competent cell to induce in vitro Immunogens CTL.In order to induce immunogens CTL, any stimulating factor can be added to cell culture.Example Such as, IL-2 is the preferred stimulating factor for CTL inductions.
In some embodiments, monitoring or the evaluation step to the immunne response of the experimenter with peptide cancer therapies Suddenly can before treatment, among and/or carry out afterwards.In general, in treatment of cancer code, repeatedly to be treated Experimenter applies immunogenic peptide.For example, it is possible to apply weekly immunogenic peptide 3-10 weeks.Correspondingly, can be in whole cancer The immunne response of experimenter is evaluated or is monitored in treatment procedure.Or, evaluate or monitoring immunne response the step of can treatment Carry out when program is completed.
According to the present invention, the induction of immunogens CTL strengthens relative to control, and show will to evaluate or diagnose is tested Immunogen immune response of the person to having applied.May include that for example, immunocompetence is thin for evaluating the suitable control of immunne response CTL induced levels when born of the same parents are not contacted with peptide, or with aminoacid sequence (for example random amino outside MPHOSPH1 peptides Acid sequence) control peptide contact when CTL induced levels.In a preferred embodiment, in sequence-specific mode The immunne response of experimenter is evaluated, method is relative immunity response between the various immunogens for being administered to experimenter.Specifically, Even if when the mixture of several MPHOSPH1 peptides is administered to experimenter, immunne response can also depend on peptide and change.At this In the case of kind, by the immunne response of the every kind of peptide of comparison, experimenter can be identified the peptide of higher response is shown to which.
XI. antibody：
Present invention also offers the antibody combined with the peptide of the present invention.Preferred antibody specificity combine the peptide of the present invention and Will not combine（Or can faint combination）Other peptides.Or, antibody can be in conjunction with the peptide of the present invention and its congener.For peptide of the present invention Antibody can be used for cancer diagnosis and prognostic assays and the imaging side science of law.Similarly, this antibody-like can be used for other cancers Treatment, diagnosis, and/or prognosis, as long as also expression or overexpression MPHOSPH1 in cancer patient.Additionally, the antibody of intracellular expression （Such as single-chain antibody）Can be used for treating the cancer for being related to MPHOSPH1 expression in treatment, the example of such cancer include but Be not limited to bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, Carcinoma of prostate, renal carcinoma and soft tissue neoplasms.
Present invention also offers panimmunity algoscopy, for detection and/or quantitative MPHOSPH1 albumen (SEQ ID NO:126) or its fragment, including by being selected from SEQ ID NO:5,14,64,73,77,79,97,103 and 120 Amino acid profile Polypeptide.Such algoscopy can include being capable of identify that for one or more and with reference to MPHOSPH1 albumen or its fragment as needed Anti- MPHOSPH1 antibody.In the linguistic context of the present invention, the anti-MPHOSPH1 antibody combined with MPHOSPH1 polypeptides preferably will identification By selected from SEQ ID NO:The polypeptide that 5,14,64,73,77,79,97,103 and 120 aminoacid sequence is constituted, and nonrecognition its Its peptide.The binding specificity of antibody can be confirmed using test is suppressed.That is, when antibody to be analyzed and total length MPHOSPH1 polypeptide Between combination any be selected from SEQ ID NO:5,14,64,73,77,79,97,103 and 120 aminoacid sequence When being suppressed in the presence of Fragment Polymorphism, it is believed that this antibody specificity combines the fragment.In the linguistic context of the present invention, this para-immunity Learn algoscopy to implement with various immunologic assay forms well known in the art, including but not limited to various types of radioimmunities are surveyed Determine method, immunochromatography technique, enzyme-linked immunosorbent assay (ELISA), enzyme linked immunological fluorimetry (ELIFA), etc..
Related immunological but non-antibody the algoscopy of the present invention also includes T cell immunogenicity determining method（Inhibition Or irritating）And MHC binding assays.In addition, the present invention also provides the cancer that can detect expression MPHOSPH1 Radioimmunological imaging method, including but not limited to using the radioscintigraphic imaging method of labeled antibody of the present invention.Such measure Method clinically can be used for express MPHOSPH1 cancer detection, monitoring and prognosis, the cancer but be not limited to bladder cancer, Breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma And soft tissue neoplasms.
Present invention also offers the antibody combined with the peptide of the present invention.The antibody of the present invention can be used in any form, Such as monoclonal or polyclonal antibody, and the anti-blood including being obtained by the peptide immune animal (such as rabbit) with the present invention Clearly, the polyclone of all kinds and monoclonal antibody and the human antibody generated by genetic recombination and humanized antibody.
The peptide for being used for obtaining antibody as antigen of the present invention can derive from any animal species, but be preferably derived from suckling Animal, such as people, mice or rat, more preferably derive from people.From people peptide can by nucleotide disclosed herein or Aminoacid sequence is obtained.
Partial peptide according to the present invention, complete protein or protein can serve as immunizing antigen.Suitable partial peptide Example include, amino (N) end of peptide for example of the present invention or carboxyl (C) terminal fragment.
Herein, antibody is defined as the protein that can be reacted with the total length of MPHOSPH1 peptides or fragment.Excellent at one In the embodiment of choosing, the antibody of the present invention will recognize have selected from SEQ ID NO:5,14,64,73,77,79,97,103 Fragment peptide with the MPHOSPH1 of 120 aminoacid sequence.Method for synthetic oligopeptide is well known in the art.After synthesis, Can optionally before using as immunogen purified peptide.In the linguistic context of the present invention, oligopeptide（Such as 9 or 10 polymers）Can be with carrier It is conjugated or connects to strengthen immunogenicity.Keyhole worm relative hemocyanin (KLH) is known as carrier.For be conjugated KLH and The method of peptide is also well known in the art.
Or, then known for the gene insertion for encoding peptide of the present invention or its fragment expression vector can be used the carrier Convert host cell described herein.Required peptide or its piece can be reclaimed from host cell outside or inside by any standard method Section, then can be used as antigen.Or, the peptide of the intact cell of expression of peptides or its lysis thing or chemosynthesis also is used as Antigen.
Any mammal of antigen immune can be used, but preferably consideration is compatible with the parental cell for cell fusion Property.Generally, the dynamic of Rodentia (Rodentia), Lagomorpha (Lagomorpha) or primatess (Primate family) can be used Thing.Cricetid includes such as mice, rat and hamster.Tu Xing sections animal includes such as rabbit.Primatess include for example narrow Nose ape (Catarrhini) (the Eastern Hemisphere monkey (old worldmonkey)) such as machin (Macaca fascicularis), perseverance River monkey (rhesus monkey), baboon (sacred baboon) and chimpanzee (chimpanzee).
It is known in the art with the method for antigen-immunized animal.Peritoneal injection or subcutaneous injection of antigens are immune sucklings The standard method of animal.In particular, can in appropriate phosphate buffered saline (PBS) (PBS), normal saline etc. dilution and Suspension antigen.If desired, antigen suspension can be mixed with appropriate standard adjuvant such as Freund (Freund) Freund's complete adjuvant, Emulsion is made, mammal is then applied to.Preferably, not exclusively help with appropriate Freund per 4-21 days administered several times thereafter The antigen of agent mixing.It is also possible to use suitable carrier carries out immunity.After immunity proceeded as above, can be examined by standard method The increase of the amount of antibody needed for blood count is clear.
Polyclonal antibody for of the present invention peptide is prepared as follows can：From the mammal (mammal for passing through immunity Through checking antibody needed for its serum to increase) collect blood, and serum is separated from blood by any conventional method.Polyclone Antibody may include the serum containing polyclonal antibody, and can detached from the serum, containing the polyclonal antibody Fraction.The affinity column that can have peptide of the present invention using such as coupling prepares immunoglobulin from the fraction for only recognizing peptide of the present invention G or M, and further using protein A or the Protein G column purification fraction.
In order to prepare for the monoclonal antibody in linguistic context of the present invention, from checking through antigen immune and as mentioned above bleeding The elevated mammal of antibody horizontal needed for clear collects immunocyte, and carries out cell fusion.Immunity for cell fusion Cell is preferably obtained from spleen.Other will include the myeloma of such as mammal with the preferred parental cell of above-mentioned immunocyte fusion Cell, more preferably has for the myeloma cell of the characteristic obtained with medicament selection fused cell.
Can be according to (the Galfre and Milstein, Methods Enzymol 73 such as known method, such as Milstein: 3-46 (1981)) method, above-mentioned immunocyte is merged with myeloma cell.
For the hybridoma obtained by cell fusion, can be (fast containing secondary Huang in standard selection medium such as HAT culture medium The culture medium of purine, aminopterin and thymidine) in culture being selected.Generally, cell culture is carried out continuously in HAT culture medium A couple of days to several weeks, the time of culture be enough to make all other cell (nonfused cell) in addition to required hybridoma dead Die.It is then possible to carry out standard limiting dilution (standard limiting dilution) to screen and clone needed for generation The hybridoma of antibody.
In addition to the above-mentioned method for hybridoma being prepared with antigen immune non-human animal, can also use peptide, expression in vitro The cell of peptide or its lysis thing immunity human lymphocyte, such as those have infected the human lymphocyte of Epstein-Barr virus.It is then possible to make Through lymphocyte and the such as U266 of the myeloma cell from the people fusions that infinitely can divide of immunity, desired to produce Hybridoma (the disclosed but unexamined Japanese patent application No.Sho 63- of the human antibody that can be combined are generated with the peptide 17688).
Subsequently gained hybridoma can be implanted into mouse peritoneal, and extract ascites.The monoclonal antibody of gained can pass through The affinity column that such as ammonium sulfate precipitation, protein A or Protein G post, DEAE ion-exchange chromatographies or coupling have peptide of the present invention carries out pure Change.The antibody of the present invention cannot be only used for the peptide of purification and the detection present invention, be also used as the agonist of peptide of the present invention and short of money The material standed for of anti-agent.
Or, immunocyte (such as through the lymphocyte of the immunity) immortality of generation antibody can be made by oncogene Change, and be used for preparing monoclonal antibody.
The monoclonal antibody for so obtaining can also be prepared by recombinant using gene engineering and (be see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, by MacMillan Publishers LTD are in Britain issuing (1990)).For example, it is possible to from the immunocyte such as hybridoma for generating antibody or through exempting from The DNA is inserted suitable carrier, and it is anti-with Prepare restructuring to import host cell by the DNA of epidemic disease lymphocyte clone encoding antibody Body.The present invention also provides recombinant antibodies prepared as described above.
The antibody of the present invention can be antibody fragment or the antibody through modification, as long as it combines one or more present invention Peptide.For example, antibody fragment can be Fab, F (ab ')2, Fv or by the Fv fragments from H chains and L chains pass through suitable joint ScFv (scFv) (Huston et al., the Proc Natl Acad Sci USA 85 being formed by connecting:5879-83 (1988)).In particular, can be by generating antibody piece with enzyme such as papain or pepsin antibody Section.Or, the gene of Encoding Antibody Fragment can be built, expression vector is inserted into, and is expressed in suitable host cell Co et al., J Immunol 152 is see, for example, (:2968-76(1994);Better and Horwitz,Methods Enzymol 178:476-96(1989);Pluckthun and Skerra,Methods Enzymol 178:497-515 (1989);Lamoyi,Methods Enzymol 121:652-63(1986);Rousseaux et al.,Methods Enzymol 121:663-9(1986);Bird and Walker,Trends Biotechnol 9:132-7(1991)).
Antibody can be by being conjugated to modify with different kinds of molecules such as Polyethylene Glycol (PEG).The invention provides passing through this The antibody of sample modification.The antibody through modifying can be obtained by chemical modification antibody.These method of modifying are this area routines 's.
Or, can with the chimeric antibody that is derived between the variable region and constant region from human antibody of non-human antibody or Humanized antibody comprising the complementary determining region (CDR) for being derived from non-human antibody, the framework region (FR) and constant region that are derived from human antibody Form come obtain the present invention antibody.This antibody-like can be prepared according to known technology.Humanization can be by with Rodents CDR or CDR sequence replace the corresponding sequence of human antibody to carry out (see, for example, Verhoeyen et al., Science 239: 1534-1536(1988)).Thus, such humanized antibody is chimeric antibody, wherein the sub-fraction of people's variable domain by from The corresponding sequence of non-human species is replaced.
Completely human antibody can also be used, such antibody is also variable comprising people in addition to people framework region and constant region Area.This antibody-like can be prepared using multiple technologies known in the art.For example, in vitro method includes that use is illustrated in phage On human antibody fragment restructuring library (such as Hoogenboom＆Winter, J.Mol.Biol.227:381(1991)).Similar , can be by human immunoglobulin gene's seat be imported transgenic animal, such as endogenous immunoglobulin Gene Partial or complete The mice of inactivation, next life human antibodies.The method is recorded in such as United States Patent (USP) Nos.6,150,584；5,545,807；5, 545,806；5,569,825；5,625,126；5,633,425；5,661,016.
Can be by the antibody purification being obtained as described above to homogeneity.For example, can be according to the separation for general protein and purification Method carries out the separation of antibody and purification.For example, it is possible to pass through suitably to select and be applied in combination column chromatography such as affinity chromatograph, mistake Filter, ultrafiltration, saltout, dialyse, sds polyacrylamide gel electrophoresis and isoelectrofocusing to be separating and separation antibody (Antibodies: A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but it is not limited thereto.Protein A post and Protein G post can serve as affinity column.Exemplary spendable protein A post Including such as Hyper D, POROS and Sepharose F.F. (Pharmacia).
The example of the suitable chromatographic technique in addition to affinity chromatograph includes such as ion-exchange chromatography, hydrophobic chromatography, gel Filtration, reversed phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization:A Laboratory Course Manual.Ed Daniel R.Marshak et al.,Cold Spring Harbor Laboratory Press(1996)).Chromatography code, such as HPLC can be carried out by liquid chromatography (LC) And FPLC.
For example, can using absorbance measuring, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), put Penetrate immunoassay (RIA) and/or immunofluorescence to measure the antigen-binding activity of antibody of the present invention.In ELISA, by this Bright antibody immobilization onboard, the peptide of the present invention is applied on the plate, then applies the sample containing desired antibody, such as Generate culture supernatants or the antibody of purification of the cell of antibody.Then apply with enzyme (such as alkali phosphatase) labelling, The second antibody of the first antibody is recognized, and plate is incubated.Then, after washing, zymolyte, such as phosphoric acid is added in plate P-nitrophenyl ester, and absorbance is measured with the antigen-binding activity of evaluate sample.Can use peptide fragment, such as C- ends or N- terminal fragments assess the binding activity of antibody as antigen.The present invention can be assessed using BIAcore (Pharmacia) The activity of antibody.
Said method is allowed by the antibody of the present invention is exposed to sample of the hypothesis containing peptide of the present invention, and is detected or surveyed The immune complex formed by antibody and peptide is measured, the peptide of the present invention is detected or measure.
Because the peptide detection or measuring method according to the present invention specific can be detected or measurement peptide, can use in this way In the various experiments using peptide.
XII. carrier and host cell：
Present invention also offers the carrier of coding peptide of the present invention and the host for being imported with the polynucleotide for encoding peptide of the present invention Cell.The carrier of the present invention can be used for the polynucleotide carrying body (carrier) in host cell as the present invention, especially DNA, for express the present invention peptide, or for apply the present invention polynucleotide for gene therapy.
When from escherichia coli as host cell and at escherichia coli (such as JM109, DH5 α, HB101 or XL1Blue) In a large amount of amplifications and when generating carrier, carrier should have " (duplication) starting point " that be suitable for expand in escherichia coli and be suitable for selecting Select the colibacillary marker gene through converting and (for example pass through medicine such as ampicillin, tetracycline, kanamycin, chlorine Mycin etc. carries out the drug resistance gene of selection).It is, for example possible to use M13 serial carriers, pUC serial carriers, pBR322, PBluescript, pCR-Script etc..In addition, as above-mentioned carrier, pGEM-T, pDIRECT and pT7 can be used for Asia Clone and extraction cDNA.When the protein of the present invention is generated using carrier, expression vector is useful.For example, be big The expression vector that expresses in enterobacteria should possess the above-mentioned feature expanded in escherichia coli.When using escherichia coli such as When JM109, DH5 α, HB101 or XL1Blue are as host cell, carrier should have can be needed for E. coli The promoter of gene, such as lacZ promoteres (Ward et al., Nature 341:544-6(1989);FASEB J 6: 2422-7 (1992)), araB promoteres (Better et al., Science 240:1041-3 (1988)), T7 promoteres etc.. In this respect, it is possible to use such as pGEX-5X-1 (Pharmacia), " QIAexpress systems " (Qiagen), pEGFP and pET (in this case, host is preferably the BL21 for expressing t7 rna polymerase) is replacing above-mentioned carrier.In addition, carrier can be with Comprising the signal sequence for peptide secretion.Instructing peptide to secrete to the Illustrative signal sequences of colibacillus periplasm has pelB signal sequences Row (Lei et al., J Bacteriol 169:4379(1987)).For the means of vector introduction target host cell are included Such as Calcium Chloride Method and electroporation.
In addition to escherichia coli, expression vector (the such as pcDNA3 for example originating from mammal can also be used And pEGF-BOS (Nucleic Acids Res 18 (17) (Invitrogen):5322 (1990)), pEF, pCDM8), be derived from elder brother The expression vector (such as " Bac-to-BAC baculovirus expression systems " (GIBCO BRL), pBacPAK8) of worm cell, be derived from plant The expression vector (such as pMH1, pMH2) of thing, the expression vector (such as pHSV, pMV, pAdexLcw) for being derived from animal viruss, source From retroviral expression vector (such as pZIpneo), be derived from yeast expression vector (such as " Pichia anomala expression reagent Box " (Invitrogen), pNV11, SP-Q01) and the expression vector (such as pPL608, pKTH50) from bacillus subtilises To generate the polypeptide of the present invention.
For expression vector in zooblast such as CHO, COS or NIH3T3 cell, carrier should be carried in the cell In carry out expressing necessary promoter, such as SV40 promoteres (Mulligan et al., Nature 277:108 (1979)), MMLV-LTR promoteres, EF1 α promoteres (Mizushima et al., Nucleic Acids Res 18:5322 (1990)), CMV promoter, etc., and be preferred for select transformant marker gene (for example by medicine (for example newly mould Element, G418) carry out the drug resistance gene of selection).Have these features known carrier example include such as pMAM, PDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
The present invention is more fully described below with reference to embodiment.But, although following material, method and embodiment may Contribute to those of ordinary skill in the art to prepare and use the particular of the present invention, they are merely intended to for illustrating The method of the present invention, therefore without limitation on the scope of the present invention.As ordinary skill thinks to recognize, and herein The similar or equivalent method of the method for description and material and material can be used for implementing or check the present invention.
Embodiment
Material and method
Cell line
T2, HLA-A*0201- positive B- Lymphoblastoids system, HLA-A*0206- positive B- lymphoblast samples Cell line, HT1376, J82, COS7 and UM-UC3 are purchased from ATCC.MKN-45 is purchased from JCRB.
The candidate for being derived from the peptide of MPHOSPH1 is selected
Using with reference to forecasting software " BIMAS " (http://www-bimas.cit.nih.gov/molbio/hla_bind) (Parker et al.,J Immunol 1994,152(1):163-75;Kuzushima et al.,Blood 2001,98 (6):1872-81) predict derived from MPHOSPH1 (GenBank Accession No:EAW63006(SEQ ID NO:40)) Combination HLA-A*0201 molecules 9 aggressiveness and 10 mer peptides.These peptides are by BioSynthesis (Lewisville, Texas) Synthesized according to standard solid-phase synthetic method, and pass through reversed-phase high-performance liquid chromatography (HPLC) purification.Respectively by analytical type HPLC and Mass spectral analyses come determine peptide purity (>And identity 90%).Peptide is dissolved in dimethyl sulfoxide with 20mg/ml and is stored in -80 ℃.
External CTL inductions
Induced as antigen-presenting cell using the dendritic cell (DC) of monocyte derived thin in vain for people is presented on Cytotoxic T lymphocyte (CTL) response of the peptide on extracellular antigen (HLA).As described in elsewhere (Nakahara S et al., Cancer Res 2003 Jul 15,63(14):4112-8) DC is produced in vitro.Specifically, for Ficoll-Paque Plus (Pharmacia) solution from Healthy Volunteers (HLA-A*0201 positive) detached PERIPHERAL BLOOD MONONUCLEAR CELL, by viscous Attached plastic tissue culture plate (Becton Dickinson) is separated, and they are enriched with as monocyte fraction. To be enriched monocytic colony containing 2% heat-inactivated autoserum (AS) AIM-V culture medium (Invitrogen) in, - 4 (R＆D of granulocyte-macrophage colony stimutaing factor (R＆D System) and 1000U/ml interleukins (IL) in 1000U/ml System cultivate in the presence of).Culture 7 days after, by the DC through cytokine induction in AIM-V culture medium in 3 micrograms/ml β With 20 micrograms/ml each synthetic peptide in 37 DEG C of impulses 3 hours in the presence of 2- microglobulins.At it on the cell that generated is apparent Cell surface on express DC correlation molecules, such as CD80, CD83, CD86 and HLA II classes（Data do not show）.Then will These DC x-ray irradiations through peptide impulse（20Gy）Inactivation, and with 1:20 ratios with CD8 the positive separating kit (Dynal) the autologous CD8+T mixing with cells obtained by positive selection.These cultures are set up in 48 orifice plates (Corning)；Per Contain 1.5X10 in 0.5ml AIM-V/2%AS culture medium in individual hole4Individual DC, 3X10 through peptide impulse5Individual CD8+T cell and 10ng/ml IL-7(R&D System).After 3 days, IL-2 (CHIRON) to final concentration 20IU/ml is supplemented to these cultures.? 7th day and the 14th day, T cell is further stimulated with the autologous DC through peptide impulse.Pass through side same as above every time Formula is preparing DC.CTL (Tanaka H et of the testing needle to the T2 cells through peptide impulse after stimulating in third round peptide at the 21st day al.,Br J Cancer 2001 Jan 5,84(1):94-9;Umano Y et al.,Br J Cancer 2001 Apr 20, 84(8):1052-7;Uchida N et al.,Clin Cancer Res 2004 Dec 15,10(24):8577-86;Suda T et al.,Cancer Sci 2006 May,97(5):411-9;Watanabe T et al.,Cancer Sci 2005 Aug,96(8):498-506).
CTL expands code
Using with Riddell et al. (1995 Oct 19,333 (16) of Walter EA et al., N Engl J Med: 1038-44;Riddell SR et al.,Nat Med 1996 Feb,2(2):The similar method of method 216-23) recorded exists CTL is expanded in culture.To 5X10 altogether4Individual CTL suspends in 25ml AIM-V/5%AS culture medium, wherein has anti-in 40ng/ml Two kinds of people's B- Lymphoblastoids systems of mitomycin C inactivations in the presence of CD3 monoclonal antibodies (Pharmingen).Open Dynamic culture one day after, adds 120IU/ml IL-2 to culture.Added to culture at the 5th day, the 8th day and the 11st day fresh The AIM-V/5%AS culture medium containing 30IU/ml IL-2 (2001 Jan 5 of Tanaka H et al., Br J Cancer, 84(1):94-9;Umano Y et al.,Br J Cancer 2001 Apr 20,84(8):1052-7;Uchida N et al.,Clin Cancer Res 2004 Dec 15,10(24):8577-86;Suda T et al.,Cancer Sci 2006 May,97(5):411-9;Watanabe T et al.,Cancer Sci 2005 Aug,96(8):498-506).
The foundation of ctl clone
In 96 round bottom microtitration plates (Nalge Nunc International) it is diluted to obtain 0.3,1 and 3 Individual CTL/ holes.In the AIM-V culture medium containing 5%AS in 150 microlitres/hole altogether, by CTL and 1x104Two kinds of individual cells/well People's B- Lymphoblastoids system, the anti-cd 3 antibodies of 30ng/ml, and the IL-2 of 125U/ml are cultivated together.Backward training in 10 days The IL-2 in 50 microlitres/hole is added in foster base reaching the IL-2 of final concentration 125U/ml.CTL activity was tested at the 14th day, and is used Same procedure amplification ctl clone (2004 Dec 15,10 of Uchida N et al., Clin Cancer Res as above (24):8577-86;Suda T et al.,Cancer Sci 2006 May,97(5):411-9;Watanabe T et al., Cancer Sci 2005 Aug,96(8):498-506).
Specific CTL activity
In order to check specific CTL activity, interferon (IFN)-γ ELISpots (ELISPOT) algoscopy is implemented With IFN-γ enzyme-linked immunosorbent assay (ELISA).Prepare the T2 (1x10 through peptide impulse4/ hole) as stimulation cell.Use Cell, CTL systems and the ctl clone that cultivates in 48 orifice plates is used as responsive cell.Regulation implement IFN-γ according to manufacturer ELISPOT algoscopys and IFN-γ ELISA algoscopys.
CTL recognizes the ability of the target cell system of endogenous expression MPHOSPH1 and HLA-A*0201
Checked the ability that ctl clone recognizes the target cell system of endogenous expression MPHOSPH1 and HLA-A*0201.To set up Ctl clone and target cell system (5X104/ hole) culture is overnight twice (twoovernight).After incubation, measured with ELISA IFN-γ in culture medium.IFN-γ ELISA is carried out according to the program of manufacturer.
Cytotoxic activity
Checked the ability that ctl clone kills the tumor cell of endogenous expression MPHOSPH1 and HLA-A*0201.Target cell The Na of (tumor cell line) with 100micro-Ci2 51CrO4(Perkin Elmer) is in CO2Labelling 1 hour in incubator.Peptide impulse Target cell by before labelling prepared by the peptide incubation 16 hours of cell and 20 micrograms/ml.To be marked with51The target of Cr is thin Born of the same parents are cleaned and are mixed with 200 microlitres of final volume in 96 hole round bottom microtitration plates with CTL.By flat board be centrifuged (800rpm, 4 points Clock) to increase contact of the cell with cell, it is placed in CO2In incubator.After incubating 4 hours, from each 50 microlitres of hole collection Clear liquid, determines radioactivity using gamma counter (PerkinElmer).Ratio is calculated according to the following formula51The percentage ratio of Cr releases is determining The percentage ratio of specific cytotoxicity：{ (cpm of the spontaneous releases of cpm of test sample release)/(cpm of maximum release is spontaneous The cpm of release) } X100.Spontaneous being released through under conditions of without effector lymphocyte individually incubate target cell to determine.Maximum is released Put obtained by target is incubated with 1N HCl.All measurements are carried out in triplicate.
Force the foundation of the cell of expression target gene and/or HLA-A0206
By PCR come amplification coding target gene open reading-frame or the cDNA of HLA-A*0206.Pcr amplification product is cloned into table Reach carrier.Plasmid transfection is entered COS7 (target bases using Lipofectamine2000 (Invitrogen) according to the code of manufacturer The negative cell line of cause and HLA-A*0206).From transfection after 2 days, harvested through the thin of transfection with Versene (Invitrogen) Born of the same parents, and the stimulation cell as CTL activity algoscopy（5X104Individual cells/well）.
CTL recognizes the ability of the target cell system of endogenous expression MPHOSPH1 and HLA-A*0206
Checked the ability that the ctl clone recognizes the target cell of endogenous expression MPHOSPH1 and HLA-A*0206.To set up CTL systems and clone with target cell system (5X104/ hole) culture is overnight twice.After incubation, measured in culture medium by ELISA IFN-γ.IFN-γ ELISA is carried out according to the program of manufacturer.
As a result
In cancer, MPHOSPH1 expression is improved
Show MPHOSPH1 using the global gene expression profile data that cDNA microarraies are obtained from various cancers (GenBank Accession No.NM_016195;SEQ ID No:125) expression in cancerous tissue compared to corresponding just Often tissue is significantly raised.8,18 cervical cancers in MPHOSPH1 expression 30 in 31 bladder cancer, 36 breast carcinoma In 18,5 in 17 cholangiocellular carcinomas, 25 in 31 CML, 6 in 11 colorectal cancers, in 14 gastric cancer 6,5 in 5 NSCLC, 7 in 7 lymphoma, 6 in 10 osteosarcoma, 7 in 22 carcinoma of prostate, (table 1) is raised significantly in 10 in 18 renal carcinomas and 21 soft tissue sarcomies.
Table 1：Compared to the ratio for normally corresponding to the case that structure observation is raised to MPHOSPH1 in cancerous tissue
Cancer/tumor Ratio
Bladder cancer 30/31
Breast carcinoma 8/36
Cervical cancer 18/18
Cholangiocellular carcinoma 5/17
CML 25/31
Colorectal cancer 6/11
Gastric cancer 6/14
NSCLC 5/5
Lymphoma 7/7
Osteosarcoma 6/10
Carcinoma of prostate 7/22
Renal carcinoma 10/18
Soft tissue sarcoma 15/21
The prediction of the HLA-A02 binding peptides derived from MPHOSPH1
With the order of high binding affinity, table 2a and 2b show that 9 aggressiveness of HLA-A02 associativities of MPHOSPH1 and 10 gathers Body peptide.47 kinds of peptides with potential HLA-A02 binding abilities altogether are selected and checked, to determine epitope peptide.
Table 2a
9 mer peptides of HLA-A02 associativities derived from MPHOSPH1
Original position Aminoacid sequence Score SEQ ID NO
575 KLLDLIEDL 1278.3 1
282 YIYDLFVPV 1096.6 2
298 KMLRLSQDV 650.5 3
218 ALLRQIKEV 591.9 4
850 FLLTIENEL 363.6 5
1108 ALSELTQGV 285.2 6
331 KLGIKHQSV 243.4 7
1689 TLQKFGDFL 218.8 8
1251 KLTDAKKQI 149.7 9
638 RLAIFKDLV 129.5 10
1467 QLTEKDSDL 87.6 11
1195 NLQDMKHLL 87.6 12
270 SVWVSFFEI 83.5 13
129 FQGCIMQPV 74.6 14
839 VLQENNEGL 73.0 15
1094 TLDVQIQHV 64.0 16
1019 AIWEECKEI 48.8 17
1696 FLQHSPSIL 40.3 18
528 DLMEDEDLV 38.8 19
406 SLLTLGKCI 38.6 20
1400 KLTNLQDEL 36.6 21
170 GILPRTLNV 35.4 22
171 ILPRTLNVL 34.2 23
786 KICSERKRV 33.5 24
880 SLSEKKNLT 30.6 25
944 LMHTKIDEL 29.6 26
1422 WLEEKMMLI 29.0 27
466 TLNVLKFSA 28.8 28
1539 KLQTEPLST 26.1 29
132 CIMQPVKDL 25.0 30
1260 KQVQKEVSV 24.7 31
1184 KLKEEITQL 24.7 32
888 TLSKEVQQI 24.0 33
280 NEYIYDLFV 23.8 34
552 LLDEDLDKT 23.4 35
461 IAYDETLNV 21.5 36
980 NLPNTQLDL 21.4 37
409 TLGKCINVL 20.1 38
175 TLNVLFDSL 19.9 39
923 KLSNEIETA 19.6 40
1389 KEHENNTDV 19.4 41
987 DLLGNDYLV 19.3 42
920 KIMKLSNEI 18.6 43
1703 ILQSKAKKI 17.7 44
512 ILNVKRATI 17.7 45
1124 KELETILET 17.7 46
453 IVNISQCYL 17.5 47
771 LICNETVEV 16.3 48
623 TLLQEREIL 15.9 49
560 TLEENKAFI 15.1 50
1415 YNADRKKWL 14.5 51
307 KGYSFIKDL 13.7 52
133 IMQPVKDLL 12.9 53
1594 KMAVKHPGC 12.6 54
365 SEMSRVIRV 11.5 55
1191 QLTNNLQDM 11.4 56
871 QIVHFQQEL 11.2 57
245 NISEFEESI 11.0 58
484 TLNSSQEKL 10.5 59
764 SLIINNKLI 10.4 60
587 LINEKKEKL 10.0 61
263 MANSIKFSV 9.525 62
1354 VLEAKLEEV 8.528 63
846 GLRAFLLTI 6.93 64
83 ILDSQTVVL 5.956 65
1562 VLDSCEVST 5.067 66
15 YVFSADPIA 3.033 67
1741 YTSEISSPI 2.733 68
959 SQISNIDLL 2.441 69
82 HILDSQTVV 2.022 70
Table 2b
10 mer peptides of HLA-A02 associativities derived from MPHOSPH1
Original position Aminoacid sequence Score SEQ ID NO
1274 KLLRiKINEL 636.3 71
551 KLLDeDLDKT 445.9 72
460 YLAYdETLNV 319.9 73
943 KLMHtKIDEL 311.8 74
262 NMANsIKFSV 291.3 75
178 VLFDsLQERL 269.9 76
770 KLICnETVEV 243.4 77
34 KLDLsHEFSL 173.5 78
407 LLTLgKCINV 118.2 79
1714 TMSSsKLSNV 115.5 80
1353 QVLEaKLEEV 104.0 81
880 SLSEkKNLTL 87.6 82
235 TLYGsLTNSL 68.4 83
1019 AIWEeCKEIV 65.4 84
552 LLDEdLDKTL 59.6 85
1093 VTLDvQIQHV 57.3 86
559 KTLEeNKAFI 42.3 87
1332 KIIEdMRMTL 42.2 88
152 GLTNsGKTYT 41.0 89
830 NIAEiEDIRV 39.2 90
586 KLINeKKEKL 36.6 91
182 SLQErLYTKM 30.6 92
1043 QQIEkLQAEV 28.9 93
870 KQIVhFQQEL 28.8 94
1318 QQYErACKDL 28.4 95
452 MIVNiSQCYL 27.5 96
923 KLSNeIETAT 26.1 97
1257 KQIKqVQKEV 24.7 98
980 NLPNtQLDLL 24.1 99
985 QLDLlGNDYL 23.0 100
1427 MMLItQAKEA 22.6 101
1523 QIMDiKPKRI 21.8 102
1484 QLVAaLEIQL 21.4 103
466 TLNVlKFSAI 19.8 104
511 KILNvKRATI 18.6 105
1340 TLEEqEQTQV 18.3 106
372 RVSElSLCDL 17.6 107
1561 VVLDsCEVST 16.8 108
309 YSFIkDLQWI 14.7 109
353 SIFTvKILQI 12.2 110
1094 TLDVqIQHVV 11.4 111
1688 GTLQkFGDFL 11.2 112
311 FIKDlQWIQV 10.7 113
1079 TLIQqLKEEL 10.5 114
1128 TILEtQKVEC 10.4 115
1487 AALEiQLKAL 10.4 116
170 GILPrTLNVL 10.2 117
503 SLDSNSNSKI 4.173 118
1107 RALSELTQGV 3.574 119
282 YIYDLFVPVS 2.216 120
160 YTFQGTEENI 1.208 121
174 RTLNVLFDSL 1.022 122
82 HILDSQTVVL 0.621 123
128 FFQGCIMQPV 0.511 124
Original position represents the amino acid residue number from the N-terminal of MPHOSPH1
Obtained by " BIMAS " in conjunction with score
The HLA-A*0201 restricted type inducing peptide CTL from MPHOSPH1 using prediction
The code described in " material and method " is used to be prepared for those CTL for the peptide for being derived from MPHOSPH1.Pass through IFN-γ ELISPOT algoscopys determine peptide-specific CTL activity (Fig. 1).Following hole number shows strong compared with control wells IFN-γ is generated：With MPHOSPH1-A02-9-850 (SEQ ID NO：5) (a) stimulates No. 7 holes, MPHOSPH1-A02-9- is used 129(SEQ ID NO：14) (b) stimulates No. 5 holes, MPHOSPH1-A02-9-846 (SEQ ID NO are used：64) (c) stimulate 5 Number hole, use MPHOSPH1-A02-10-460 (SEQ ID NO：73) (d) stimulates No. 2 holes, MPHOSPH1-A02-10-770 is used (SEQ ID NO：77) (e) stimulates No. 1 hole, MPHOSPH1-A02-10-407 (SEQ ID NO are used：79) (f) stimulate No. 1 Hole, use MPHOSPH1-A02-10-923 (SEQ ID NO：97) (g) stimulates No. 4 holes, MPHOSPH1-A02-10-1484 is used (SEQ ID NO：103) (h) stimulates No. 5 holes and with MPHOSPH1-A02-10-282 (SEQ ID NO：120) (i) stimulates No. 8 holes.On the other hand, stimulated with other peptides in table 2a and 2b and fail to detect specific CTL activity, although these peptides have Possible binding activity to HLA-A*0201.As typical negative data instance, from MPHOSPH1-A02-9-575 (SEQ ID NO：1) CTL that (j) stimulates does not observe that specificity IFN-γ is generated.As a whole, these result promptings are selected 10 kinds of peptides derived from MPHOSPH1 can induce strong CTL.
The foundation of CTL systems and clone for MPHOSPH1 derived peptide
Will be with MPHOSPH1-A02-9-850 (SEQ ID NO：5) (a) stimulates No. 7 holes, MPHOSPH1-A02-9- is used 129(SEQ ID NO：14) (b) stimulates No. 5 holes, MPHOSPH1-A02-9-846 (SEQ ID NO are used：64) (c) stimulate 5 Number hole, use MPHOSPH1-A02-10-460 (SEQ ID NO：73) (d) stimulates No. 2 holes, MPHOSPH1-A02-10-770 is used (SEQ ID NO：77) (e) stimulates No. 1 hole, MPHOSPH1-A02-10-407 (SEQ ID NO are used：79) (f) stimulate No. 1 Hole, use MPHOSPH1-A02-10-923 (SEQ ID NO：97) (g) stimulates No. 4 holes, MPHOSPH1-A02-10-1484 is used (SEQ ID NO：103) (h) stimulates No. 5 holes and with MPHOSPH1-A02-10-282 (SEQ ID NO：120) (i) stimulates Show that the cell of peptide-specific CTL activity is expanded by the detection of IFN-γ ELISPOT algoscopys in No. 8 holes, and set up CTL systems (Fig. 2).The CTL activity that these CTL systems are determined by IFN-γ ELISA.With the T2 cell phases without peptide impulse Than these CTL systems show strong IFN-γ for the T2 cells through corresponding peptides impulse and generate.Additionally, from these CTL systems such as Ctl clone is set up by limiting dilution described in " material and method ", and is determined from these CTL gram by IFN-γ ELISA The grand IFN-γ for the T2 cells with corresponding peptides impulse is generated.From MPHOSPH1-A02-9-MPHOSPH1850 (SEQ ID NO：5) (a), MPHOSPH1-A02-9-846 (SEQ ID NO：64) (b), MPHOSPH1-A02-10-460 (SEQ ID NO： 73) (c), MPHOSPH1-A02-10-770 (SEQ ID NO：77) (d) and MPHOSPH1-A02-10-282 (SEQ ID NO： 120) ctl clone that (e) stimulates has measured strong IFN-γ and has generated (Fig. 3).
Specific CTL activity for the target cell of expression MPHOSPH1 and HLA-A*0201
For the ctl clone for building up, the ability of the target cell of recognition expression MPHOSPH1 and HLA-A*0201 molecule is checked. With MPHOSPH1-A02-10-282 (SEQ ID NO：120) ctl clone for stimulating is for expression MPHOSPH1 and HLA-A*0201 The J82 cells of the two show strong CTL activity.On the other hand, MPHOSPH1 but do not express HLA-A*0201's for expression HT1376 cells or expression HLA-A*0201 but do not express the T2 cells of MPHOSPH1 and be not detected by significant specific CTL and live Property (Fig. 4).Therefore, the data are clearly illustrated, MPHOSPH1-A02-10-282 (SEQ ID NO：120) peptide by endogenous plus Work is simultaneously rendered on target cell together with HLA-A*0201 molecules, and recognized by CTL.
Cytotoxic activity for the tumor cell line of expression MPHOSPH1 and HLA-A*0201
For the ctl clone for building up, the tumor that they recognize and kill expression MPHOSPH1 and HLA-A*0201 is checked The ability of cell line.With MPHOSPH1-A02-10-282 (SEQ ID NO：120) ctl clone for stimulating is for expression The UMUC-3 cells of both MPHOSPH1 and HLA-A*0201 show strong cytotoxic activity.On the other hand, for expression MPHOSPH1 but do not express the MKN45 cells and expression HLA-A*0201 of HLA-A*0201 but do not express the T2 of MPHOSPH1 The ctl clone of cell is not detected by significant specific CTL activity (Fig. 5).These results show derived from MPHOSPH1 MPHOSPH1-A02-10-282 (SEQ ID NO：120) peptide may can be applied to the tumor with expression MPHOSPH1 The cancer vaccine of patient.
Specific CTL activity for the target cell of expression MPHOSPH1 and HLA-A*0206
For for MPHOSPH1-A02-10-282 (SEQ ID NO：120) the CTL systems that peptide is set up, checked which and recognize The ability of the target cell of expression MPHOSPH1 and HLA-A*0206 molecules.Prepare with total length MPHOSPH1 and HLA-A*0206 genes The COS7 cells (specific model of the target cell of expression MPHOSPH1 and HLA-A*0206 genes) of the two transfection are used as stimulation Cell, and the COS7 cells transfected with total length MPHOSPH1 or HLA-A*0206 are used as control.In figure 6, use MPHOSPH1-A02-10-282(SEQ ID NO：120) ctl clone for stimulating is for expression MPHOSPH1 and HLA-A*0206 bis- The COS7 cells of person show strong CTL activity.On the other hand, the significant specific CTL activity for control is not detected by.Cause This, these data clearly illustrate MPHOSPH1-A02-10-282 (SEQ ID NO：120) peptide is processed and and HLA- by endogenous A*0206 molecules are rendered on target cell together, and are recognized by CTL.
Homology analysis to antigenic peptides
Through MPHOSPH1-A02-9-850 (SEQ ID NO：5), MPHOSPH1-A02-9-129 (SEQ ID NO：14), MPHOSPH1-A02-9-846(SEQ ID NO：64), MPHOSPH1-A02-10-460 (SEQ ID NO：73), MPHOSPH1- A02-10-770(SEQ ID NO：77), MPHOSPH1-A02-10-407 (SEQ ID NO：79), MPHOSPH1-A02-10- 923(SEQ ID NO：97), MPHOSPH1-A02-10-1484 (SEQ ID NO：And MPHOSPH1-A02-10-282 103) (SEQ ID NO：120) CTL for stimulating shows significant and special CTL activity.This result is likely due to MPHOSPH1-A02-9-850(SEQ ID NO：MPHOSPH15), MPHOSPH1-A02-9-129 (SEQ ID NO： MPHOSPH114), MPHOSPH1-A02-9-846 (SEQ ID NO：MPHOSPH164), MPHOSPH1-A02-10-460 (SEQ ID NO：MPHOSPH173), MPHOSPH1-A02-10-770 (SEQ ID NO：77), MPHOSPH1-A02-10-407 (SEQ ID NO：79), MPHOSPH1-A02-10-923 (SEQ ID NO：97), MPHOSPH1-A02-10-1484 (SEQ ID NO： 103) with MPHOSPH1-A02-10-282 (SEQ ID NO：120) sequence and the other known human immune system sensitization of making Derived from molecule, peptide is homologous.For ruled it out, using BLAST algorithm (http:// Www.ncbi.nlm.nih.gov/blast/blast.cgi these peptide sequences) are used to implement homology point as search terms Analysis, does not have the sequence for finding there is notable homology.The result of homology analysis indicates MPHOSPH1-A02-9-850 (SEQ ID NO：MPHOSPH15), MPHOSPH1-A02-9-129 (SEQ ID NO：MPHOSPH114), MPHOSPH1-A02-9-846 (SEQ ID NO：MPHOSPH164), MPHOSPH1-A02-10-460 (SEQ ID NO：MPHOSPH173), MPHOSPH1-A02-10- 770(SEQ ID NO：77), MPHOSPH1-A02-10-407 (SEQ ID NO：79), MPHOSPH1-A02-10-923 (SEQ ID NO：97), MPHOSPH1-A02-10-1484 (SEQ ID NO：103) with MPHOSPH1-A02-10-282 (SEQ ID NO： 120) sequence is unique, and therefore, as far as we know, these molecules produce the undesirable immunology to some irrelevant molecules The probability very little of response.
In a word, the new epitope peptides of the HLA-A*0201 derived from MPHOSPH1 of identification herein may be useful in cancer immunity Therapy field.
Industrial applicability
The invention provides new TAA, is particularly derived from the new TAA of MPHOSPH1, they are inducible strong and specific Anti-tumor immune response, and can be applicable to extensive cancer types.Such TAA for its as MPHOSPH1 correlation disease Disease, such as cancer, more specifically, bladder cancer, breast carcinoma, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, gastric cancer, The further exploitation of the peptide vaccine of NSCLC, lymphoma, osteosarcoma, carcinoma of prostate, renal carcinoma and soft tissue neoplasms provides foundation.
Although describe in detail the present invention herein in reference to its specific embodiment, it is to be appreciated that above description Substantially be exemplary and explanatory, and be intended to illustrate the present invention and its preferred embodiment.Via normal experiment, this Art personnel will readily recognize that, can carry out various change and modification to the present invention, without departing from the essence of the present invention God and scope, the border and scope of the present invention are limited by claim below and its equivalent.

Claims (18)

1. one kind is by SEQ ID NO:The detached peptide of 120 compositions.
2. a kind of detached polynucleotide, its encode the peptide of claim 1.
3. a kind of compositionss for inducing CTL, peptide of the wherein described compositionss comprising claim 1, or coding for said peptides Polynucleotide.
4. a kind of compositionss of the APC for induction with CTL inducibilities, wherein described compositionss are comprising claim 1 Peptide, or the polynucleotide of one or more coding for said peptides.
5. a kind of pharmaceutical composition, which includes at least one active component being selected from the group：
The peptide of (a) claim 1,
The polynucleotide of the peptide of (b) one or more coding claim 1；
C () presents the APC of the have the right peptide of requirement 1 and the complex of HLA antigens for one or more in its surface；
D () presents the allochthon of the have the right peptide of requirement 1 and the complex of HLA antigens for one or more in its surface；With
E () one or more identification presents the cell of the have the right peptide of requirement 1 and the complex of HLA antigens in its surface CTL.
6. the pharmaceutical composition of claim 5, for being used for treatment and/or prophylaxis of cancer in experimenter, or induces for cancer Immunne response.
7. the pharmaceutical composition of claim 6, wherein described pharmaceutical composition are formulated as being applied to HLA antigens for HLA-A2's Experimenter.
8. a kind of in vitro method of the antigen-presenting cell (APC) for induction with CTL inducibilities, methods described includes selecting From the following group the step of：
A () makes APC contact with the peptide of claim 1, and
B the polynucleotide for encoding the peptide of claim 1 are imported APC by ().
9. a kind of in vitro method for inducing CTL, methods described include the step of being selected from the group：
A CD8 positive T cells are had HLA antigens common with the APC of the complex of the peptide of claim 1 with presenting in its surface by () Culture；
B () CD8 positive T cells and presentation in its surface are had the allochthon of HLA antigens and the complex of the peptide of claim 1 Co-culture；With
C polynucleotide or coding that () imports two φt cell receptor (TCR) subunits of coding in CD8 positive T cells are every Multiple polynucleotide of one TCR subunit, wherein described TCR can be in conjunction with the HLA antigens and power presented on cell surface Profit requires the complex of 1 peptide.
10. a kind of detached APC, the APC present the complex of HLA antigens and the peptide of claim 1 in its surface.
A kind of 11. APC, its are induced by the method for claim 8.
12. a kind of detached CTL, which recognizes to present in its surface has HLA antigens thin with the complex of the peptide of claim 1 Born of the same parents.
A kind of 13. CTL, its are induced by the method for claim 9.
14. active component are formulated as the purposes in the pharmaceutical composition for treatment and/or prophylaxis of cancer, the activity in preparation Composition is selected from：
The peptide of (a) claim 1；
The polynucleotide of the peptide of (b) one or more coding claim 1；
C () presents the APC of the have the right peptide of requirement 1 and the complex of HLA antigens for one or more in its surface；
D () presents the allochthon of the have the right peptide of requirement 1 and the complex of HLA antigens for one or more in its surface；With
E () one or more identification presents the cell of the have the right peptide of requirement 1 and the complex of HLA antigens in its surface CTL.
The polynucleotide of the peptide or coding for said peptides of 15. claim 1 are being prepared for induction pin in experimenter in need To the purposes in the pharmaceutical composition of the immunne response of cancer.
A kind of 16. carriers, its include the nucleotide sequence of the peptide of coding claim 1.
A kind of 17. host cells, its carrier conversion or transfection through claim 16.
A kind of 18. diagnostic kits, its include the polynucleotide of the peptide or coding for said peptides of claim 1.
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